Nanophotonic structures with optical surface modes for tunable spin current generation

P. Shilina; D. O. Ignatyeva; P. O. Kapralov; S. K. Sekatskii; M. Nur-E-Alam et al. 

We propose a novel type of photonic-crystal (PC)-based nanostructures for efficient and tunable optically-induced spin current generation via the spin Seebeck and inverse spin Hall effects. It has been experimentally demonstrated that optical surface modes localized at the PC surface covered by ferromagnetic layer and materials with giant spin-orbit coupling (SOC) notably increase the efficiency of the optically-induced spin current generation, and provides its tunability by modifying the light wavelength or angle of incidence. Up to 100% of the incident light power can be transferred to heat within the SOC layer and, therefore, to the spin current. Importantly, the high efficiency becomes accessible even for ultra-thin SOC layers. Moreover, the surface patterning of the PC-based spintronic nanostructure allows for the local generation of spin currents at the pattern scales rather than the diameter of the laser beam.

Sensing of Surface and Bulk Refractive Index Using Magnetophotonic Crystal with Hybrid Magneto-Optical Response

D. Ignatyeva; P. Kapralov; P. Golovko; P. Shilina; A. Khramova et al. 

We propose an all-dielectric magneto-photonic crystal with a hybrid magneto-optical response that allows for the simultaneous measurements of the surface and bulk refractive index of the analyzed substance. The approach is based on two different spectral features of the magneto-optical response corresponding to the resonances in p- and s-polarizations of the incident light. Angular spectra of p-polarized light have a step-like behavior near the total internal reflection angle which position is sensitive to the bulk refractive index. S-polarized light excites the TE-polarized optical Tamm surface mode localized in a submicron region near the photonic crystal surface and is sensitive to the refractive index of the near-surface analyte. We propose to measure a hybrid magneto-optical intensity modulation of p-polarized light obtained by switching the magnetic field between the transverse and polar configurations. The transversal component of the external magnetic field is responsible for the magneto-optical resonance near total internal reflection conditions, and the polar component reveals the resonance of the Tamm surface mode. Therefore, both surface- and bulk-associated features are present in the magneto-optical spectra of the p-polarized light. View Full-Text Keywords: optical sensor; photonic crystal; surface optical wave; magneto-optics; transverse magneto-optical Kerr effect; Faraday effect

Nanomotion Detection-Based Rapid Antibiotic Susceptibility Testing

S. Kasas; A. Malovichko; M. I. Villalba; M. E. Vela; O. Yantorno et al. 

Rapid antibiotic susceptibility testing (AST) could play a major role in fighting multidrug-resistant bacteria. Recently, it was discovered that all living organisms oscillate in the range of nanometers and that these oscillations, referred to as nanomotion, stop as soon the organism dies. This finding led to the development of rapid AST techniques based on the monitoring of these oscillations upon exposure to antibiotics. In this review, we explain the working principle of this novel technique, compare the method with current ASTs, explore its application and give some advice about its implementation. As an illustrative example, we present the application of the technique to the slowly growing and pathogenic Bordetella pertussis bacteria.

Environmental Control of Amyloid Polymorphism by Modulation of Hydrodynamic Stress

J. Zhou; L. Venturelli; L. Keiser; S. K. Sekatskii; F. Gallaire et al. 

The phenomenon of amyloid polymorphism is a key feature of protein aggregation. Unravelling this phenomenon is of great significance for understanding the underlying molecular mechanisms associated with neurodegenerative diseases and for the development of amyloid-based functional biomaterials. However, the understanding of the molecular origins and the physicochemical factors modulating amyloid polymorphs remains challenging. Herein, we demonstrate an association between amyloid polymorphism and environmental stress in solution, induced by an air/water interface in motion. Our results reveal that low-stress environments produce heterogeneous amyloid polymorphs, including twisted, helical, and rod-like fibrils, whereas high-stress conditions generate only homogeneous rod-like fibrils. Moreover, high environmental stress converts twisted fibrils into rodlike fibrils both in-pathway and after the completion of mature amyloid formation. These results enrich our understanding of the environmental origin of polymorphism of pathological amyloids and shed light on the potential of environmentally controlled fabrication of homogeneous amyloid biomaterials for biotechnological applications.

Single Particle Analysis for High-Resolution 2D Electron Crystallography

R. Righetto; H. Stahlberg 

Alterations in Sub-Axonal Architecture Between Normal Aging and Parkinson’s Diseased Human Brains Using Label-Free Cryogenic X-ray Nanotomography

H. T. Tran; E. H. R. Tsai; A. J. Lewis; T. Moors; J. G. J. M. Bol et al. 

High-resolution cryo-EM structure of urease from the pathogen Yersinia enterocolitica

R. D. Righetto; L. Anton; R. Adaixo; R. P. Jakob; J. Zivanov et al. 

Urease converts urea into ammonia and carbon dioxide and makes urea available as a nitrogen source for all forms of life except animals. In human bacterial pathogens, ureases also aid in the invasion of acidic environments such as the stomach by raising the surrounding pH. Here, we report the structure of urease from the pathogen Yersinia enterocolitica at 2 Å resolution from cryo-electron microscopy. Y. enterocolitica urease is a dodecameric assembly of a trimer of three protein chains, ureA, ureB and ureC. The high data quality enables detailed visualization of the urease bimetal active site and of the impact of radiation damage. The obtained structure is of sufficient quality to support drug development efforts.

Structural investigation of ACE2 dependent disassembly of the trimeric SARS-CoV-2 Spike glycoprotein

D. Ni; K. Lau; F. Lehmann; A. Fränkl; D. Hacker et al. 

The human membrane protein Angiotensin-converting enzyme 2 (hACE2) acts as the main receptor for host cells invasion of the new coronavirus SARS-CoV-2. The viral surface glycoprotein Spike binds to hACE2, which triggers virus entry into cells. As of today, the role of hACE2 for virus fusion is not well understood. Blocking the transition of Spike from its prefusion to post-fusion state might be a strategy to prevent or treat COVID-19. Here we report a single particle cryo-electron microscopy analysis of SARS-CoV-2 trimeric Spike in presence of the human ACE2 ectodomain. The binding of purified hACE2 ectodomain to Spike induces the disassembly of the trimeric form of Spike and a structural rearrangement of its S1 domain to form a stable, monomeric complex with hACE2. This observed hACE2 dependent dissociation of the Spike trimer suggests a mechanism for the therapeutic role of recombinant soluble hACE2 for treatment of COVID-19.

Architecture of the centriole cartwheel‐containing region revealed by cryo‐electron tomography

N. Klena; M. Le Guennec; A. Tassin; H. van den Hoek; P. S. Erdmann et al. 

Centrioles are evolutionarily conserved barrels of microtubule triplets that form the core of the centrosome and the base of the cilium. While the crucial role of the proximal region in centriole biogenesis has been well documented, its native architecture and evolutionary conservation remain relatively unexplored. Here, using cryo‐electron tomography of centrioles from four evolutionarily distant species, we report on the architectural diversity of the centriole’s proximal cartwheel‐bearing region. Our work reveals that the cartwheel central hub is constructed from a stack of paired rings with cartwheel inner densities inside. In both Paramecium and Chlamydomonas, the repeating structural unit of the cartwheel has a periodicity of 25 nm and consists of three ring pairs, with 6 radial spokes emanating and merging into a single bundle that connects to the microtubule triplet via the D2‐rod and the pinhead. Finally, we identified that the cartwheel is indirectly connected to the A‐C linker through the triplet base structure extending from the pinhead. Together, our work provides unprecedented evolutionary insights into the architecture of the centriole proximal region, which underlies centriole biogenesis.

Single particle cryo‐EM of the complex between interphotoreceptor retinoid‐binding protein and a monoclonal antibody

A. E. Sears; S. Albiez; S. Gulati; B. Wang; P. Kiser et al. 

Interphotoreceptor retinoid‐binding protein (IRBP) is a highly expressed protein secreted by rod and cone photoreceptors that has major roles in photoreceptor homeostasis as well as retinoid and polyunsaturated fatty acid transport between the neural retina and retinal pigment epithelium. Despite two crystal structures reported on fragments of IRBP and decades of research, the overall structure of IRBP and function within the visual cycle remain unsolved. Here, we studied the structure of native bovine IRBP in complex with a monoclonal antibody (mAb5) by cryo‐electron microscopy, revealing the tertiary and quaternary structure at sufficient resolution to clearly identify the complex components. Complementary mass spectrometry experiments revealed the structure and locations of N‐linked carbohydrate post‐translational modifications. This work provides insight into the structure of IRBP, displaying an elongated, flexible three‐dimensional architecture not seen among other retinoid‐binding proteins. This work is the first step in elucidation of the function of this enigmatic protein.

Structural basis of Focal Adhesion Kinase activation on lipid membranes

I. Acebrón; R. D. Righetto; C. Schoenherr; S. Buhr; P. Redondo et al. 

Focal adhesion kinase (FAK) is a key component of the membrane proximal signaling layer in focal adhesion complexes, regulating important cellular processes, including cell migration, proliferation, and survival. In the cytosol, FAK adopts an autoinhibited state but is activated upon recruitment into focal adhesions, yet how this occurs or what induces structural changes is unknown. Here, we employ cryo‐electron microscopy to reveal how FAK associates with lipid membranes and how membrane interactions unlock FAK autoinhibition to promote activation. Intriguingly, initial binding of FAK to the membrane causes steric clashes that release the kinase domain from autoinhibition, allowing it to undergo a large conformational change and interact itself with the membrane in an orientation that places the active site toward the membrane. In this conformation, the autophosphorylation site is exposed and multiple interfaces align to promote FAK oligomerization on the membrane. We show that interfaces responsible for initial dimerization and membrane attachment are essential for FAK autophosphorylation and resulting cellular activity including cancer cell invasion, while stable FAK oligomerization appears to be needed for optimal cancer cell proliferation in an anchorage‐independent manner. Together, our data provide structural details of a key membrane bound state of FAK that is primed for efficient autophosphorylation and activation, hence revealing the critical event in integrin mediated FAK activation and signaling at focal adhesions.

Protease-activation using anti-idiotypic masks enables tumor specificity of a folate receptor 1-T cell bispecific antibody

M. Geiger; K-G. Stubenrauch; J. Sam; W. F. Richter; G. Jordan et al. 

T-cell bispecific antibodies (TCBs) crosslink tumor and T-cells to induce tumor cell killing. While TCBs are very potent, on-target off-tumor toxicity remains a challenge when selecting targets. Here, we describe a protease-activated anti-folate receptor 1 TCB (Prot-FOLR1-TCB) equipped with an anti-idiotypic anti-CD3 mask connected to the anti-CD3 Fab through a tumor protease-cleavable linker. The potency of this Prot- FOLR1-TCB is recovered following protease-cleavage of the linker releasing the anti-idiotypic anti-CD3 scFv. In vivo, the Prot-FOLR1-TCB mediates antitumor efficacy comparable to the parental FOLR1-TCB whereas a noncleavable control Prot-FOLR1-TCB is inactive. In contrast, killing of bronchial epithelial and renal cortical cells with low FOLR1 expression is prevented compared to the parental FOLR1-TCB. The findings are confirmed for mesothelin as alternative tumor antigen. Thus, masking the anti-CD3 Fab fragment with an anti-idiotypic mask and cleavage of the mask by tumor-specific proteases can be applied to enhance specificity and safety of TCBs.

The Contorsbody, an antibody format for agonism: design, structure, and function

G. J. Georges; S. Dengl; A. Bujotzek; F. Hesse; J. A. Fischer et al. 

The careful design of the antibody architecture is becoming more and more important, especially when the purpose is agonism. We present the design of a novel antibody format that is able to promote receptor dimerization and induce signal transduction resulting in cell proliferation. Mono-specific bivalent Y-shape IgGs made of two light chains and two heavy chains are engineered into single chain dimers of two modified heavy chains, resulting in the fixation of the two Fab fragments along the Fc dimerizing moiety. By this, an antagonist of the Her-receptor family, Trastuzumab, is re-formatted into an agonist by simply incorporating the original binding motif into a different geometrically and sterically constrained conformation. This novel format, named Contorsbody, retains antigen binding properties of the parental IgGs and can be produced by standard technologies established for recombinant IgGs. Structural analyses using molecular dynamics and electron microscopy are described to guide the initial design and to confirm the Contorsbody as a very compact molecule, respectively. Contorsbodies show increased rigidity compared to IgGs and their Fab moieties are positioned parallel and adjacent to each other. This geometry has an increased potential to trigger cell surface antigen or receptor ‘cis’ dimerization without ‘trans’-bridging of cells or mere receptor blockade.

Cryo-EM, X-ray diffraction, and atomistic simulations reveal determinants for the formation of a supramolecular myelin-like proteolipid lattice

S. Ruskamo; O. C. Krokengen; J. Kowal; T. Nieminen; M. Lehtimaki et al. 

Myelin protein P2 is a peripheral membrane protein of the fatty acid–binding protein family that functions in the formation and maintenance of the peripheral nerve myelin sheath. Several P2 gene mutations cause human Charcot–Marie–Tooth neuropathy, but the mature myelin sheath assembly mechanism is unclear. Here, cryo-EM of myelin-like proteolipid multilayers revealed an ordered 3D lattice of P2 molecules between stacked lipid bilayers, visualizing supramolecular assembly at the myelin major dense line. The data disclosed that a single P2 layer is inserted between two bilayers in a tight intermembrane space of ~3 nm, implying direct interactions between P2 and two membrane surfaces. X-ray diffraction from P2-stacked bicelle multilayers revealed lateral protein organization, and surface mutagenesis of P2 coupled with structure–function experiments revealed a role for both the portal region of P2 and its opposite face in membrane interactions. Atomistic molecular dynamics simulations of P2 on model membrane surfaces suggested that Arg88 is critical for P2–membrane interactions, in addition to the helical lid domain. Negatively charged lipid headgroups stably anchored P2 on the myelin-like bilayer surface. Membrane binding may be accompanied by opening of the P2 β-barrel structure and ligand exchange with the apposing bilayer. Our results provide an unprecedented view into an ordered, multilayered biomolecular membrane system induced by the presence of a peripheral membrane protein from human myelin. This is an important step towards deciphering the 3D assembly of a mature myelin sheath at the molecular level.

New insights on the structure of alpha-synuclein fibrils using cryo-electron microscopy

R. Guerrero-Ferreira; L. Kovacik; D. Ni; H. Stahlberg 

Fibrils of alpha-synuclein are significant components of cellular inclusions associated with several neuropathological disorders including Parkinson’s disease, multiple system atrophy and dementia with Lewy bodies. In recent years, technological advances in the field of transmission electron microscopy and image processing have made it possible to solve the structure of alpha-synuclein fibrils at high resolution. This review discusses the results of structural studies using cryo-electron microscopy, which revealed that in-vitro produced fibrils vary in diameter from 5 nm for single-protofilament fibrils, to 10 nm for two-protofilament fibrils. In addition, the atomic models hint at contributions of the familial Parkinson’s disease mutation sites to inter-protofilament interaction and the locations where post-translational modifications take place. Here, we propose a nomenclature system that allows identifying the existing alpha-synuclein polymorphs and that will allow to incorporate additional high-resolution structures determined in the future.

Grayscale e-beam lithography: Effects of a delayed development for well-controlled 3D patterning

T. Mortelmans; D. Kazazis; V. A. Guzenko; C. Padeste; T. Braun et al. 

Grayscale electron beam lithography (g-EBL) is a fabrication technique that allows for tunable control of resist topography. In most cases, the height of the structures is in the submicron regime. Here, we present an extensive experimental characterization of the post electron beam exposure behavior of poly(methyl methacrylate) (PMMA) 950 K for grayscale structuring with several micrometers in height. The obtained results show that the development depth for the same electron dose is dependent on the time between exposure and development. This dependence becomes more prominent at higher exposure doses. Additionally, it was found that a post-exposure bake influences the dose-response behavior of the resist material and, therefore, also the obtained three-dimensional (3D) structure. This work paves the way for well-controlled 3D micrometer structuring via g-EBL.

A helical inner scaffold provides a structural basis for centriole cohesion

M. Le Guennec; N. Klena; D. Gambarotto; M. H. Laporte; A-M. Tassin et al. 

The ninefold radial arrangement of microtubule triplets (MTTs) is the hallmark of the centriole, a conserved organelle crucial for the formation of centrosomes and cilia. Although strong cohesion between MTTs is critical to resist forces applied by ciliary beating and the mitotic spindle, how the centriole maintains its structural integrity is not known. Using cryo–electron tomography and subtomogram averaging of centrioles from four evolutionarily distant species, we found that MTTs are bound together by a helical inner scaffold covering ~70% of the centriole length that maintains MTTs cohesion under compressive forces. Ultrastructure Expansion Microscopy (U-ExM) indicated that POC5, POC1B, FAM161A, and Centrin-2 localize to the scaffold structure along the inner wall of the centriole MTTs. Moreover, we established that these four proteins interact with each other to form a complex that binds microtubules. Together, our results provide a structural and molecular basis for centriole cohesion and geometry.

Two new polymorphic structures of human full-length alpha-synuclein fibrils solved by cryo-electron microscopy

R. Guerrero-Ferreira; N. M. Taylor; A-A. Arteni; P. Kumari; D. Mona et al. 

Intracellular inclusions rich in alpha-synuclein are a hallmark of several neuropathological diseases including Parkinson’s disease (PD). Previously, we reported the structure of alpha-synuclein fibrils (residues 1–121), composed of two protofibrils that are connected via a densely-packed interface formed by residues 50–57 (Guerrero-Ferreira, eLife 218;7:e36402). We here report two new polymorphic atomic structures of alpha-synuclein fibrils termed polymorphs 2a and 2b, at 3.0 Å and 3.4 Å resolution, respectively. These polymorphs show a radically different structure compared to previously reported polymorphs. The new structures have a 10 nm fibril diameter and are composed of two protofilaments which interact via intermolecular salt-bridges between amino acids K45, E57 (polymorph 2a) or E46 (polymorph 2b). The non-amyloid component (NAC) region of alpha-synuclein is fully buried by previously non-described interactions with the N-terminus. A hydrophobic cleft, the location of familial PD mutation sites, and the nature of the protofilament interface now invite to formulate hypotheses about fibril formation, growth and stability.

Imaging of post-mortem human brain tissue using electron and X-ray microscopy

A. J. Lewis; C. Genoud; M. Pont; W. D. J. van de Berg; S. Frank et al. 

Electron microscopy imaging of post-mortem human brain (PMHB) comes with a unique set of challenges due to numerous parameters beyond the researcher’s control. Nevertheless, the wealth of information provided by the ultrastructural analysis of PMHB is proving crucial in our understanding of neurodegenerative diseases. This review highlights the importance of such studies and covers challenges, limitations and recent developments in the application of current EM imaging, including cryo-ET and correlative hybrid techniques, on PMHB.

DuoMab: a novel CrossMab-based IgG-derived antibody format for enhanced antibody-dependent cell-mediated cytotoxicity

C. Sustmann; S. Dickopf; J. T. Regula; H. Kettenberger; M. Molhoj et al. 

High specificity accompanied with the ability to recruit immune cells has made recombinant therapeutic antibodies an integral part of drug development. Here we present a generic approach to generate two novel IgG-derived antibody formats that are based on a modification of the CrossMab technology. MoAbs harbor two heavy chains (HCs) resulting in one binding entity and one fragment crystallizable region (Fc), whereas DuoMabs are composed of four HCs harboring two binding entities and two Fc regions linked at a disulfide-bridged hinge. The latter bivalent format is characterized by avidity-enhanced target cell binding while simultaneously increasing the ‘Fc-load’ on the surface. DuoMabs were shown to be producible in high yield and purity and bind to surface cells with affinities comparable to IgGs. The increased Fc load directed at the surface of target cells by DuoMabs modulates their antibody-dependent cell-mediated cytotoxicity competency toward target cells, making them attractive for applications that require or are modulated by FcR interactions.

Diverse roles of TssA-like proteins in the assembly of bacterial type VI secretion systems

J. P. Schneider; S. Nazarov; R. Adaixo; M. Liuzzo; P. D. Ringel et al. 

Protein translocation by the bacterial type VI secretion system (T6SS) is driven by a rapid contraction of a sheath assembled around a tube with associated effectors. Here, we show that TssA-like or TagA-like proteins with a conserved N-terminal domain and varying C-terminal domains can be grouped into at least three distinct classes based on their role in sheath assembly. The proteins of the first class increase speed and frequency of sheath assembly and form a stable dodecamer at the distal end of a polymerizing sheath. The proteins of the second class localize to the cell membrane and block sheath polymerization upon extension across the cell. This prevents excessive sheath polymerization and bending, which may result in sheath destabilization and detachment from its membrane anchor and thus result in failed secretion. The third class of these proteins localizes to the baseplate and is required for initiation of sheath assembly. Our work shows that while various proteins share a conserved N-terminal domain, their roles in T6SS biogenesis are fundamentally different.

Differential Visual Proteomics: Enabling the Proteome-Wide Comparison of Protein Structures of Single-Cells

A. Syntychaki; L. Rima; C. Schmidli; T. Stohler; A. Bieri et al. 

Proteins are involved in all tasks of life, and their characterization is essential to understand the underlying mechanisms of biological processes. We present a method called “differential visual proteomics” geared to study proteome-wide structural changes of proteins and protein-complexes between a disturbed and an undisturbed cell or between two cell populations. To implement this method, the cells are lysed and the lysate is prepared in a lossless manner for single-particle electron microscopy (EM). The samples are subsequently imaged in the EM. Individual particles are computationally extracted from the images and pooled together, while keeping track of which particle originated from which specimen. The extracted particles are then aligned and classified. A final quantitative analysis of the particle classes found identifies the particle structures that differ between positive and negative control samples. The algorithm and a graphical user interface developed to perform the analysis and to visualize the results were tested with simulated and experimental data. The results are presented, and the potential and limitations of the current implementation are discussed. We envisage the method as a tool for the untargeted profiling of the structural changes in the proteome of single-cells as a response to a disturbing force.

Microfluidic protein isolation and sample preparation for high-resolution cryo-EM

C. Schmidli; S. Albiez; L. Rima; R. Righetto; I. Mohammed et al. 

High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousand to a few million protein particles required for structure determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than 1 mu L of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens more opportunities for the isolation of sensitive, endogenous protein complexes.

Cryo-EM structure of the rhodopsin-G alpha i-beta gamma complex reveals binding of the rhodopsin C-terminal tail to the g beta subunit

C-J. Tsai; J. Marino; R. Adaixo; F. Pamulal; J. Muehle et al. 

One of the largest membrane protein families in eukaryotes are G protein-coupled receptors (GPCRs). GPCRs modulate cell physiology by activating diverse intracellular transducers, prominently heterotrimeric G proteins. The recent surge in structural data has expanded our understanding of GPCR-mediated signal transduction. However, many aspects, including the existence of transient interactions, remain elusive. We present the cryo-EM structure of the light-sensitive GPCR rhodopsin in complex with heterotrimeric Gi. Our density map reveals the receptor C-terminal tail bound to the G beta subunit of the G protein, providing a structural foundation for the role of the C-terminal tail in GPCR signaling, and of G beta as scaffold for recruiting G alpha subunits and G protein-receptor kinases. By comparing available complexes, we found a small set of common anchoring points that are G protein-subtype specific. Taken together, our structure and analysis provide new structural basis for the molecular events of the GPCR signaling pathway.

Lewy pathology in Parkinson’s disease consists of crowded organelles and lipid membranes

S. H. Shahmoradian; A. J. Lewis; C. Genoud; J. Hench; T. E. Moors et al. 

Parkinson’s disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Key neuropathological hallmarks are Lewy bodies and Lewy neurites: neuronal inclusions immunopositive for the protein alpha-synuclein. In-depth ultrastructural analysis of Lewy pathology is crucial to understanding pathogenesis of this disease. Using correlative light and electron microscopy and tomography on postmortem human brain tissue from Parkinson’s disease brain donors, we identified alpha-synuclein immunopositive Lewy pathology and show a crowded environment of membranes therein, including vesicular structures and dysmorphic organelles. Filaments interspersed between the membranes and organelles were identifiable in many but not all alpha-synuclein inclusions. Crowding of organellar components was confirmed by stimulated emission depletion (STED)-based super-resolution microscopy, and high lipid content within alpha-synuclein immunopositive inclusions was corroborated by confocal imaging, Fourier-transform coherent anti-Stokes Raman scattering infrared imaging and lipidomics. Applying such correlative high-resolution imaging and biophysical approaches, we discovered an aggregated protein-lipid compartmentalization not previously described in the Parkinsons’ disease brain.

Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy

C. Claus; C. Ferrara; W. Xu; J. Sam; S. Lang et al. 

Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic anti-4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fc gamma receptor-mediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAP-4-1BBL (RG7826) and CD19-4-1BBL. In the presence of a T cell receptor signal, they provide potent T cell costimulation strictly dependent on tumor antigen-mediated hyperclustering without systemic activation by Fc gamma R binding. We could show targeting of FAP-4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer-bearing rhesus monkey. Combination of FAP-4-1BBL with tumor antigen-targeted T cell bispecific (TCB) molecules in human tumor samples led to increased IFN-gamma and granzyme B secretion. Further, combination of FAP- or CD19-4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral accumulation of activated effector CD8(+) T cells. FAP- and CD19-4-1BBL thus represent an off-the-shelf combination immunotherapy without requiring genetic modification of effector cells for the treatment of solid and hematological malignancies.

Cryo-EM structures of the pore-forming A subunit from the Yersinia entomophaga ABC toxin

S. J. Piper; L. Brillault; R. Rothnagel; T. I. Croll; J. K. Box et al. 

ABC toxins are pore-forming virulence factors produced by pathogenic bacteria. YenTcA is the pore-forming and membrane binding A subunit of the ABC toxin YenTc, produced by the insect pathogen Yersinia entomophaga. Here we present cryo-EM structures of YenTcA, purified from the native source. The soluble pre-pore structure, determined at an average resolution of 4.4 A, reveals a pentameric assembly that in contrast to other characterised ABC toxins is formed by two TcA-like proteins (YenA1 and YenA2) and decorated by two endochitinases (Chi1 and Chi2). We also identify conformational changes that accompany membrane pore formation by visualising YenTcA inserted into liposomes. A clear outward rotation of the Chi1 subunits allows for access of the protruding translocation pore to the membrane. Our results highlight structural and functional diversity within the ABC toxin subfamily, explaining how different ABC toxins are capable of recognising diverse hosts.

Retrieving high-resolution information from disordered 2D crystals by single-particle cryo-EM

R. D. Righetto; N. Biyani; J. Kowal; M. Chami; H. Stahlberg 

Electron crystallography can reveal the structure of membrane proteins within 2D crystals under close-to-native conditions. High-resolution structural information can only be reached if crystals are perfectly flat and highly ordered. In practice, such crystals are difficult to obtain. Available image unbending algorithms correct for disorder, but only perform well on images of non-tilted, flat crystals, while out-of-plane distortions are not addressed. Here, we present an approach that employs single-particle refinement procedures to locally unbend crystals in 3D. With this method, density maps of the MloK1 potassium channel with a resolution of 4 A were obtained from images of 2D crystals that do not diffract beyond 10 A. Furthermore, 3D classification allowed multiple structures to be resolved, revealing a series of MloK1 conformations within a single 2D crystal. This conformational heterogeneity explains the poor diffraction observed and is related to channel function. The approach is implemented in the FOCUS package.

Supramolecular architectures of molecularly thin yet robust free-standing layers

M. Moradi; N. L. Opara; L. G. Tulli; C. Waeckerlin; S. J. Dalgarno et al. 

Stable, single-nanometer thin, and free-standing two-dimensional layers with controlled molecular architectures are desired for several applications ranging from (opto-)electronic devices to nanoparticle and single-biomolecule characterization. It is, however, challenging to construct these stable single molecular layers via self-assembly, as the cohesion of those systems is ensured only by in-plane bonds. We herein demonstrate that relatively weak noncovalent bonds of limited directionality such as dipole-dipole (-CN center dot center dot center dot NC-) interactions act in a synergistic fashion to stabilize crystalline monomolecular layers of tetrafunctional calixarenes. The monolayers produced, demonstrated to be freestanding, display a well-defined atomic structure on the single-nanometer scale and are robust under a wide range of conditions including photon and electron radiation. This work opens up new avenues for the fabrication of robust, single-component, and free-standing layers via bottom-up self-assembly.

Cryo-EM structure of phosphodiesterase 6 reveals insights into the allosteric regulation of type I phosphodiesterases

S. Gulati; K. Palczewski; A. Engel; H. Stahlberg; L. Kovacik 

Cyclic nucleotide phosphodiesterases (PDEs) work in conjunction with adenylate/guanylate cyclases to regulate the key second messengers of G protein-coupled receptor signaling. Previous attempts to determine the full-length structure of PDE family members at high-resolution have been hindered by structural flexibility, especially in their linker regions and N- and C-terminal ends. Therefore, most structure-activity relationship studies have so far focused on truncated and conserved catalytic domains rather than the regulatory domains that allosterically govern the activity of most PDEs. Here, we used single-particle cryo-electron microscopy to determine the structure of the full-length PDE6 alpha beta 2 gamma complex. The final density map resolved at 3.4 angstrom reveals several previously unseen structural features, including a coiled N-terminal domain and the interface of PDE6 gamma subunits with the PDE6 alpha beta heterodimer. Comparison of the PDE6 alpha beta 2 gamma complex with the closed state of PDE2A sheds light on the conformational changes associated with the allosteric activation of type I PDEs.

Molecular structure and function of myelin protein P0 in membrane stacking

A. Raasakka; S. Ruskamo; J. Kowal; H. Han; A. Baumann et al. 

Compact myelin forms the basis of nerve insulation essential for higher vertebrates. Dozens of myelin membrane bilayers undergo tight stacking, and in the peripheral nervous system, this is partially enabled by myelin protein zero (P0). Consisting of an immunoglobulin (Ig)-like extracellular domain, a single transmembrane helix, and a cytoplasmic extension (P0ct), P0 harbours an important task in ensuring the integrity of compact myelin in the extracellular compartment, referred to as the intraperiod line. Several disease mutations resulting in peripheral neuropathies have been identified for P0, reflecting its physiological importance, but the arrangement of P0 within the myelin ultrastructure remains obscure. We performed a biophysical characterization of recombinant P0ct. P0ct contributes to the binding affinity between apposed cytoplasmic myelin membrane leaflets, which not only results in changes of the bilayer properties, but also potentially involves the arrangement of the Iglike domains in a manner that stabilizes the intraperiod line. Transmission electron cryomicroscopy of native full-length P0 showed that P0 stacks lipid membranes by forming antiparallel dimers between the extracellular Ig-like domains. The zipper-like arrangement of the P0 extracellular domains between two membranes explains the double structure of the myelin intraperiod line. Our results contribute to the understanding of PNS myelin, the role of P0 therein, and the underlying molecular foundation of compact myelin stability in health and disease.

TDP-43 extracted from frontotemporal lobar degeneration subject brains displays distinct aggregate assemblies and neurotoxic effects reflecting disease progression rates

F. Laferriere; Z. Maniecka; M. Perez-Berlanga; M. Hruska-Plochan; L. Gilhespy et al. 

Accumulation of abnormally phosphorylated TDP-43 (pTDP-43) is the main pathology in affected neurons of people with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Morphological diversity and neuroanatomical distribution of pTDP-43 accumulations allowed classification of FTLD cases into at least four subtypes, which are correlated with clinical presentations and genetic causes. To understand the molecular basis of this heterogeneity, we developed SarkoSpin, a new method for biochemical isolation of pathological TDP-43. By combining SarkoSpin with mass spectrometry, we revealed proteins beyond TDP-43 that become abnormally insoluble in a disease subtype-specific manner. We show that pTDP-43 extracted from brain forms stable assemblies of distinct densities and morphologies that are associated with disease subtypes. Importantly, biochemically extracted pTDP-43 assemblies showed differential neurotoxicity and seeding that were correlated with disease duration of FTLD subjects. Our data are consistent with the notion that disease heterogeneity could originate from alternate pathological TDP-43 conformations, which are reminiscent of prion strains.

Cerebral Corpora amylacea are dense membranous labyrinths containing structurally preserved cell organelles

P. P. Navarro; C. Genoud; D. Castano-Diez; A. Graff-Meyer; A. J. Lewis et al. 

Corpora amylacea are cell-derived structures that appear physiologically in the aged human brain. While their histological identification is straightforward, their ultrastructural composition and microenvironment at the nanoscale have remained unclear so far, as has their relevance to aging and certain disease states that involve the sequestration of toxic cellular metabolites. Here, we apply correlative serial block-face scanning electron microscopy and transmission electron tomography to gain three-dimensional insight into the ultrastructure and surrounding microenvironment of cerebral Corpora amylacea in the human brainstem and hippocampal region. We find that cerebral Corpora amylacea are composed of dense labyrinth-like sheets of lipid membranes, contain vesicles as well as morphologically preserved mitochondria, and are in close proximity to blood vessels and the glymphatic system, primarily within the cytoplasm of perivascular glial cells. Our results clarify the nature of cerebral Corpora amylacea and provide first hints on how they may arise and develop in the aging brain.

Self-Assembly of a Designed Nucleoprotein Architecture through Multimodal Interactions

R. H. Subramanian; S. J. Smith; R. G. Alberstein; J. B. Bailey; L. Zhang et al. 

The co-self-assembly of proteins and nucleic acids (NAs) produces complex biomolecular machines (e.g., ribosomes and telomerases) that represent some of the most daunting targets for biomolecular design. Despite significant advances in protein and DNA or RNA nanotechnology, the construction of artificial nucleoprotein complexes has largely been limited to cases that rely on the NA-mediated spatial organization of protein units, rather than a cooperative interplay between protein-and NA-mediated interactions that typify natural nucleoprotein assemblies. We report here a structurally well-defined synthetic nucleoprotein assembly that forms through the synergy of three types of intermolecular interactions: Watson-Crick base pairing, NA-protein interactions, and protein-metal coordination. The fine thermodynamic balance between these interactions enables the formation of a crystalline architecture under highly specific conditions.

Cryo-EM structures of a human ABCG2 mutant trapped in ATP-bound and substrate-bound states

I. Manolaridis; S. M. Jackson; N. M. I. Taylor; J. Kowal; H. Stahlberg et al. 

ABCG2 is a transporter protein of the ATP-binding-cassette (ABC) family that is expressed in the plasma membrane in cells of various tissues and tissue barriers, including the blood-brain, blood-testis and maternal-fetal barriers(1-4). Powered by ATP, it translocates endogenous substrates, affects the pharmacokinetics of many drugs and protects against a wide array of xenobiotics, including anti-cancer drugs(5-12). Previous studies have revealed the architecture of ABCG2 and the structural basis of its inhibition by small molecules and antibodies(13,14). However, the mechanisms of substrate recognition and ATP-driven transport are unknown. Here we present high-resolution cryo-electron microscopy (cryo-EM) structures of human ABCG2 in a substrate-bound pre-translocation state and an ATP-bound post-translocation state. For both structures, we used a mutant containing a glutamine replacing the catalytic glutamate (ABCG2(EQ)), which resulted in reduced ATPase and transport rates and facilitated conformational trapping for structural studies. In the substrate-bound state, a single molecule of estrone-3-sulfate (E1S) is bound in a central, hydrophobic and cytoplasm-facing cavity about halfway across the membrane. Only one molecule of E1S can bind in the observed binding mode. In the ATP-bound state, the substrate-binding cavity has collapsed while an external cavity has opened to the extracellular side of the membrane. The ATP-induced conformational changes include rigid-body shifts of the transmembrane domains, pivoting of the nucleotide-binding domains (NBDs), and a change in the relative orientation of the NBD subdomains. Mutagenesis and in vitro characterization of transport and ATPase activities demonstrate the roles of specific residues in substrate recognition, including a leucine residue that forms a ‘plug’ between the two cavities. Our results show how ABCG2 harnesses the energy of ATP binding to extrude E1S and other substrates, and suggest that the size and binding affinity of compounds are important for distinguishing substrates from inhibitors.

Inner-membrane GspF of the bacterial type II secretion system is a dimeric adaptor mediating pseudopilus biogenesis

W. Van Putte; T. De Vos; W. Van Den Broeck; H. Stahlberg; M. Kudryashev et al. 

The type II secretion system (T2SS), a protein complex spanning the bacterial envelope, is pivotal to bacterial pathogenicity. Central to T2SS function is the extrusion of protein cargos from the periplasm into the extracellular environment mediated by a pseudopilus and motorized by a cytosolic ATPase. GspF, an inner-membrane component of T2SS has long been considered to be a key player in this process, yet the structural basis of its role had remained elusive. Here, we employed single-particle electron microscopy based on XcpS (GspF) from the T2SS of pathogenic P. aeruginosa stabilized by a nanobody, to show that XcpS adopts a dimeric structure mediated by its transmembrane helices. This assembly matches in terms of overall organization and dimensions the basal inner-membrane cassette of a T2SS machinery. Thus, GspF is poised to serve as an adaptor involved in the mediation of propeller-like torque generated by the motor ATPase to the secretion pseudopilus.

An iris diaphragm mechanism to gate a cyclic nucleotide-gated ion channel

A. Marchesi; X. Gao; R. Adaixo; J. Rheinberger; H. Stahlberg et al. 

Cyclic nucleotide-gated (CNG) ion channels are non-selective cation channels key to signal transduction. The free energy difference of cyclic-nucleotide (cAMP/cGMP) binding/unbinding is translated into mechanical work to modulate the open/closed probability of the pore, i.e., gating. Despite the recent advances in structural determination of CNG channels, the conformational changes associated with gating remain unknown. Here we examine the conformational dynamics of a prokaryotic homolog of CNG channels, SthK, using high-speed atomic force microscopy (HS-AFM). HS-AFM of SthK in lipid bilayers shows that the CNBDs undergo dramatic conformational changes during the interconversion between the resting (apo and cGMP) and the activated (cAMP) states: the CNBDs approach the membrane and splay away from the 4-fold channel axis accompanied by a clockwise rotation with respect to the pore domain. We propose that these movements may be converted by the C-linker to pull the pore helices open in an iris diaphragm-like mechanism.

Structure of a PSI-LHCI-cyt b(6)f supercomplex in Chlamydomonas reinhardtii promoting cyclic electron flow under anaerobic conditions

J. Steinbeck; I. L. Ross; R. Rothnagel; P. Gaebelein; S. Schulze et al. 

Photosynthetic linear electron flow (LEF) produces ATP and NADPH, while cyclic electron flow (CEF) exclusively drives photophosphorylation to supply extra ATP. The fine-tuning of linear and cyclic electron transport levels allows photosynthetic organisms to balance light energy absorption with cellular energy requirements under constantly changing light conditions. As LEF and CEF share many electron transfer components, a key question is how the same individual structural units contribute to these two different functional modes. Here, we report the structural identification of a photosystem I (PSI)-light harvesting complex I (LHCI)-cytochrome (cyt) b(6)f supercomplex isolated from the unicellular alga Chlamydomonas reinhardtii under anaerobic conditions, which induces CEF. This provides strong evidence for the model that enhanced CEF is induced by the formation of CEF supercomplexes, when stromal electron carriers are reduced, to generate additional ATP. The additional identification of PSI-LHCI-LHCII complexes is consistent with recent findings that both CEF enhancement and state transitions are triggered by similar conditions, but can occur independently from each other. Single molecule fluorescence correlation spectroscopy indicates a physical association between cyt b(6)f and fluorescent chlorophyll containing PSI-LHCI supercomplexes. Single particle analysis identified top-view projections of the corresponding PSI-LHCI-cyt b(6)f supercomplex. Based on molecular modeling and mass spectrometry analyses, we propose a model in which dissociation of LHCA2 and LHCA9 from PSI supports the formation of this CEF supercomplex. This is supported by the finding that a Delta lhca2 knockout mutant has constitutively enhanced CEF.

Protocols for Subtomogram Averaging of Membrane Proteins in the Dynamo Software Package

P. P. Navarro; H. Stahlberg; D. Castano-Diez 

Cryo-electron tomography allows low-resolution three-dimensional (3D) viewing of cellular organelles and macromolecular complexes present as multiple copies within a tomogram. These structures are computationally extracted and averaged in order to obtain high-resolution 3D structures, and provide a map of their spatial distribution and interaction with their biological microenvironment. To do so, we apply the user-friendly Dynamo software package on a tomographic data set. Dynamo acts as a modular toolbox adaptable to different biological scenarios, allowing a custom designed pipeline for subtomogram averaging. Here, we use as a textbook example the mitochondrial docking site of the positive-strand RNA Flock house nodavirus (FHV) to describe how Dynamo coordinates several basic steps in the subtomogram averaging workflow. Our framework covers specific strategies to deal with additional issues in subtomogram averaging as tomographic data management, 3D surface visualization, automatic assignment of asymmetry and inherent loss of Fourier information in presence of preferential views.

Demonstration of femtosecond X-ray pump X-ray probe diffraction on protein crystals

N. L. Opara; I. Mohacsi; M. Makita; D. Castano-Diez; A. Diaz et al. 

The development of X-ray free-electron lasers (XFELs) has opened the possibility to investigate the ultrafast dynamics of biomacromolecules using X-ray diffraction. Whereas an increasing number of structures solved by means of serial femtosecond crystallography at XFELs is available, the effect of radiation damage on protein crystals during ultrafast exposures has remained an open question. We used a splitand-delay line based on diffractive X-ray optics at the Linac Coherent Light Source XFEL to investigate the time dependence of X-ray radiation damage to lysozyme crystals. For these tests, crystals were delivered to the X-ray beam using a fixed-target approach. The presented experiments provide probe signals at eight different delay times between 19 and 213 femtoseconds after a single pump event, thereby covering the time-scales relevant for femtosecond serial crystallography. Even though significant impact on the crystals was observed at long time scales after exposure with a single X-ray pulse, the collected diffraction data did not show significant signal reduction that could be assigned to beam damage on the crystals in the sampled time window and resolution range. This observation is in agreement with estimations of the applied radiation dose, which in our experiment was clearly below the values expected to cause damage on the femtosecond time scale. The experiments presented here demonstrate the feasibility of time-resolved pump-multiprobe X-ray diffraction experiments on protein crystals. (C) 2018 Author(s).

Miniaturized Sample Preparation for Transmission Electron Microscopy

C. Schmidli; L. Rima; S. A. Arnold; T. Stohler; A. Syntychaki et al. 

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-andplunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for “visual proteomics,” or “lossless” total sample preparation for quantitative analysis of complex samples.

Cryo-EM structure of alpha-synuclein fibrils

R. Guerrero-Ferreira; N. M. I. Taylor; D. Mona; P. Ringler; M. E. Lauer et al. 

Parkinson’s disease is a progressive neuropathological disorder that belongs to the class of synucleinopathies, in which the protein alpha-synuclein is found at abnormally high concentrations in affected neurons. Its hallmark are intracellular inclusions called Lewy bodies and Lewy neurites. We here report the structure of cytotoxic alpha-synuclein fibrils (residues 1 – 121), determined by cryo-electron microscopy at a resolution of 3.4 angstrom. Two protofilaments form a polar fibril composed of staggered beta-strands. The backbone of residues 38 to 95, including the fibril core and the non-amyloid component region, are well resolved in the EM map. Residues 50 – 57, containing three of the mutation sites associated with familial synucleinopathies, form the interface between the two protofilaments and contribute to fibril stability. A hydrophobic cleft at one end of the fibril may have implications for fibril elongation, and invites for the design of molecules for diagnosis and treatment of synucleinopathies.

Structural basis for regulation of human acetyl-CoA carboxylase

M. Hunkeler; A. Hagmann; E. Stuttfeld; M. Chami; Y. Guri et al. 

Acetyl-CoA carboxylase catalyses the ATP-dependent carboxylation of acetyl-CoA, a rate-limiting step in fatty acid biosynthesis(1,2). Eukaryotic acetyl-CoA carboxylases are large, homodimeric multienzymes. Human acetyl-CoA carboxylase occurs in two isoforms: the metabolic, cytosolic ACC1, and ACC2, which is anchored to the outer mitochondrial membrane and controls fatty acid beta-oxidatio(1,3). ACC1 is regulated by a complex interplay of phosphorylation, binding of allosteric regulators and protein-protein interactions, which is further linked to filament formation(1,4-8). These filaments were discovered in vitro and in vivo 50 years ago(7,9,10), but the structural basis of ACC1 polymerization and regulation remains unknown. Here, we identify distinct activated and inhibited ACC1 filament forms. We obtained cryo-electron microscopy structures of an activated filament that is allosterically induced by citrate (ACC-citrate), and an inactivated filament form that results from binding of the BRCT domains of the breast cancer type 1 susceptibility protein (BRCA1). While non polymeric ACC1 is highly dynamic, filament formation locks ACC1 into different catalytically competent or incompetent conformational states. This unique mechanism of enzyme regulation via large-scale conformational changes observed in ACC1 has potential uses in engineering of switchable biosynthetic systems. Dissecting the regulation of acetyl-CoA carboxylase opens new paths towards counteracting upregulation of fatty acid biosynthesis in disease.

Femtosecond X-ray coherent diffraction of aligned amyloid fibrils on low background graphene

C. Seuring; K. Ayyer; E. Filippaki; M. Barthelmess; J-N. Longchamp et al. 

Here we present a new approach to diffraction imaging of amyloid fibrils, combining a freestanding graphene support and single nanofocused X-ray pulses of femtosecond duration from an X-ray free-electron laser. Due to the very low background scattering from the graphene support and mutual alignment of filaments, diffraction from tobacco mosaic virus (TMV) filaments and amyloid protofibrils is obtained to 2.7 A and 2.4 A resolution in single diffraction patterns, respectively. Some TMV diffraction patterns exhibit asymmetry that indicates the presence of a limited number of axial rotations in the XFEL focus. Signal-to-noise levels from individual diffraction patterns are enhanced using computational alignment and merging, giving patterns that are superior to those obtainable from synchrotron radiation sources. We anticipate that our approach will be a starting point for further investigations into unsolved structures of filaments and other weakly scattering objects.

Structural basis of small-molecule inhibition of human multidrug transporter ABCG2

S. M. Jackson; I. Manolaridis; J. Kowal; M. Zechner; N. M. I. Taylor et al. 

ABCG2 is an ATP-binding cassette (ABC) transporter that protects tissues against xenobiotics, affects the pharmacokinetics of drugs and contributes to multidrug resistance. Although many inhibitors and modulators of ABCG2 have been developed, understanding their structure-activity relationship requires high-resolution structural insight. Here, we present cryo-EM structures of human ABCG2 bound to synthetic derivatives of the fumitremorgin C-related inhibitor Ko143 or the multidrug resistance modulator tariquidar. Both compounds are bound to the central, inward-facing cavity of ABCG2, blocking access for substrates and preventing conformational changes required for ATP hydrolysis. The high resolutions allowed for de novo building of the entire transporter and also revealed tightly bound phospholipids and cholesterol interacting with the lipid-exposed surface of the transmembrane domains (TMDs). Extensive chemical modifications of the Ko143 scaffold combined with in vitro functional analyses revealed the details of ABCG2 interactions with this compound family and provide a basis for the design of novel inhibitors and modulators.

Image processing techniques for high-resolution structure determination from badly ordered 2D crystals

N. Biyani; S. Scherer; R. D. Righetto; J. Kowal; M. Chami et al. 

2D electron crystallography can be used to study small membrane proteins in their native environment. Obtaining highly ordered 2D crystals is difficult and time-consuming. However, 2D crystals diffracting to only 10-12 angstrom can be prepared relatively conveniently in most cases. We have developed image-processing algorithms allowing to generate a high resolution 3D structure from cryo-electron crystallography images of badly ordered crystals. These include movie-mode unbending, refinement over sub-tiles of the images in order to locally refine the sample tilt geometry, implementation of different CTF correction schemes, and an iterative method to apply known constraints in the real and reciprocal space to approximate amplitudes and phases in the so-called missing cone regions. These algorithms applied to a dataset of the potassium channel MloK1 show significant resolution improvements to better than 5 angstrom.

Miniaturizing EM Sample Preparation: Opportunities, Challenges, and “Visual Proteomics”

S. A. Arnold; S. A. Muller; C. Schmidli; A. Syntychaki; L. Rima et al. 

This review compares and discusses conventional versus miniaturized specimen preparation methods for transmission electron microscopy (TEM). The progress brought by direct electron detector cameras, software developments and automation have transformed transmission cryo-electron microscopy (cryo-EM) and made it an invaluable high-resolution structural analysis tool. In contrast, EM specimen preparation has seen very little progress in the last decades and is now one of the main bottlenecks in cryo-EM. Here, we discuss the challenges faced by specimen preparation for single particle EM, highlight current developments, and show the opportunities resulting from the advanced miniaturized and microfluidic sample grid preparation methods described, such as visual proteomics and time-resolved cryo-EM studies.

Structure of a zosuquidar and UIC2-bound human-mouse chimeric ABCB1

A. Alam; R. Kung; J. Kowal; R. A. McLeod; N. Tremp et al. 

The multidrug transporter ABCB1 (P-glycoprotein) is an ATP-binding cassette transporter that has a key role in protecting tissues from toxic insult and contributes to multidrug extrusion from cancer cells. Here, we report the near-atomic resolution cryo-EM structure of nucleotide-free ABCB1 trapped by an engineered disulfide cross-link between the nucleotide-binding domains (NBDs) and bound to the antigen-binding fragment of the human-specific inhibitory antibody UIC2 and to the third-generation ABCB1 inhibitor zosuquidar. Our structure reveals the transporter in an occluded conformation with a central, enclosed, inhibitor-binding pocket lined by residues from all transmembrane (TM) helices of ABCB1. The pocket spans almost the entire width of the lipid membrane and is occupied exclusively by two closely interacting zosuquidar molecules. The external, conformational epitope facilitating UIC2 binding is also visualized, providing a basis for its inhibition of substrate efflux. Additional cryo-EM structures suggest concerted movement of TM helices from both halves of the transporters associated with closing the NBD gap, as well as zosuquidar binding. Our results define distinct recognition interfaces of ABCB1 inhibitory agents, which may be exploited for therapeutic purposes.

3D correlative electron microscopy reveals continuity of Brucella-containing vacuoles with the endoplasmic reticulum

J. Sedzicki; T. Tschon; S. H. Low; K. Willemart; K. N. Goldie et al. 

Entry of the facultative intracellular pathogen Brucella into host cells results in the formation of endosomal Brucella-containing vacuoles (eBCVs) that initially traffic along the endocytic pathway. eBCV acidification triggers the expression of a type IV secretion system that translocates bacterial effector proteins into host cells. This interferes with lysosomal fusion of eBCVs and supports their maturation to replicative Brucella-containing vacuoles (rBCVs). Bacteria replicate in rBCVs to large numbers, eventually occupying most of the cytoplasmic volume. As rBCV membranes tightly wrap each individual bacterium, they are constantly being expanded and remodeled during exponential bacterial growth. rBCVs are known to carry endoplasmic reticulum (ER) markers; however, the relationship of the vacuole to the genuine ER has remained elusive. Here, we have reconstructed the 3-dimensional ultrastructure of rBCVs and associated ER by correlative structured illumination microscopy (SIM) and focused ion beam/scanning electron microscopic tomography (FIB/SEM). Studying B. abortus-infected HeLa cells and trophoblasts derived from B. melitensis-infected mice, we demonstrate that rBCVs are complex and interconnected compartments that are continuous with neighboring ER cisternae, thus supporting a model that rBCVs are extensions of genuine ER.

Cryo-EM reconstruction of Type VI secretion system baseplate and sheath distal end

S. Nazarov; J. P. Schneider; M. Brackmann; K. N. Goldie; H. Stahlberg et al. 

The bacterial Type VI secretion system (T6SS) assembles from three major parts: a membrane complex that spans inner and outer membranes, a baseplate, and a sheath-tube polymer. The baseplate assembles around a tip complex with associated effectors and connects to the membrane complex by TssK. The baseplate assembly initiates sheath-tube polymerization, which in some organisms requires TssA. Here, we analyzed both ends of isolated non-contractile Vibrio cholerae sheaths by cryo-electron microscopy. Our analysis suggests that the baseplate, solved to an average 8.0 angstrom resolution, is composed of six subunits of TssE/F-2/G and the baseplate periphery is decorated by six TssK trimers. The VgrG/PAAR tip complex in the center of the baseplate is surrounded by a cavity, which may accommodate up to similar to 450 kDa of effector proteins. The distal end of the sheath, resolved to an average 7.5 angstrom resolution, shows sixfold symmetry; however, its protein composition is unclear. Our structures provide an important step toward an atomic model of the complete T6SS assembly.

High-Resolution Cryoelectron Microscopy Structure of the Cyclic Nucleotide-Modulated Potassium Channel MloK1 in a Lipid Bilayer

J. Kowal; N. Biyani; M. Chami; S. Scherer; A. J. Rzepiela et al. 

Eukaryotic cyclic nucleotide-modulated channels perform their diverse physiological roles by opening and closing their pores to ions in response to cyclic nucleotide binding. We here present a structural model for the cyclic nucleotide-modulated potassium channel homolog from Mesorhizobium loti, MloK1, determined from 2D crystals in the presence of lipids. Even though crystals diffract electrons to only similar to 10 angstrom, using cryoelectron microscopy ( cryo-EM) and recently developed computational methods, we have determined a 3D map of full-length MloK1 in the presence of cyclic AMP ( cAMP) at similar to 4.5 angstrom isotropic 3D resolution. The structure provides a clear picture of the arrangement of the cyclic nucleotide-binding domains with respect to both the pore and the putative voltage sensor domains when cAMP is bound, and reveals a potential gating mechanism in the context of the lipid-embedded channel.

MRCZ – A file format for cryo-TEM data with fast compression

R. A. McLeod; R. D. Righetto; A. Stewart; H. Stahlberg 

The introduction of fast CMOS detectors is moving the field of transmission electron microscopy into the computer science field of big data. Automated data pipelines control the instrument and initial processing steps which imposes more onerous data transfer and archiving requirements. Here we conduct a technical demonstration whereby storage and read/write times are improved 10 x at a dose rate of 1 e(-)/pix/frame for data from a Gatan K2 direct-detection device by combination of integer decimation and lossless compression. The example project is hosted at github.com/em-MRCZ and released under the BSD license.

Cryo-EM structure of the extended type VI secretion system sheath-tube complex

J. Wang; M. Brackmann; D. Castano-Diez; M. Kudryashev; K. N. Goldie et al. 

The bacterial type VI secretion system (T6SS) uses contraction of a long sheath to quickly thrust a tube with associated effectors across membranes of eukaryotic and bacterial cells(1-5). Only limited structural information is available about the inherently unstable precontraction state of the T6SS. Here, we obtain a 3.7 angstrom resolution structure of a non-contractile sheath-tube complex using cryo-electron microscopy and show that it resembles the extended T6SS inside Vibrio cholerae cells. We build a pseudo-atomic model of the complete sheath-tube assembly, which provides a mechanistic understanding of coupling sheath contraction with pushing and rotating the inner tube for efficient target membrane penetration. Our data further show that sheath contraction exposes a buried recognition domain to specifically trigger the disassembly and recycling of the T6SS sheath by the cognate ATP-dependent unfoldase ClpV.

Cryo-EM analysis of homodimeric full-length LRRK2 and LRRK1 protein complexes

K. Sejwal; M. Chami; H. Remigy; R. Vancraenenbroeck; W. Sibran et al. 

Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein implicated in the pathogenesis of both familial and sporadic Parkinson’s disease (PD), and currently one of the most promising therapeutic targets for drug design in Parkinson’s disease. In contrast, LRRK1, the closest homologue to LRRK2, does not play any role in PD. Here, we use cryo-electron microscopy (cryo-EM) and single particle analysis to gain structural insight into the full-length dimeric structures of LRRK2 and LRRK1. Differential scanning fluorimetry-based screening of purification buffers showed that elution of the purified LRRK2 protein in a high pH buffer is beneficial in obtaining high quality cryo-EM images. Next, analysis of the 3D maps generated from the cryo-EM data show 16 and 25 angstrom resolution structures of full length LRRK2 and LRRK1, respectively, revealing the overall shape of the dimers with two-fold symmetric orientations of the protomers that is closely similar between the two proteins. These results suggest that dimerization mechanisms of both LRRKs are closely related and hence that specificities in functions of each LRRK are likely derived from LRRK2 and LRRK1’s other biochemical functions. To our knowledge, this study is the first to provide 3D structural insights in LRRK2 and LRRK1 dimers in parallel.

Three-Dimensional Imaging of Biological Tissue by Cryo X-Ray Ptychography

S. H. Shahmoradian; E. H. R. Tsai; A. Diaz; M. Guizar-Sicairos; J. Raabe et al. 

High-throughput three-dimensional cryogenic imaging of thick biological specimens is valuable for identifying biologically-or pathologically-relevant features of interest, especially for subsequent correlative studies. Unfortunately, high-resolution imaging techniques at cryogenic conditions often require sample reduction through sequential physical milling or sectioning for sufficient penetration to generate each image of the 3-D stack. This study represents the first demonstration of using ptychographic hard X-ray tomography at cryogenic temperatures for imaging thick biological tissue in a chemically-fixed, frozen-hydrated state without heavy metal staining and organic solvents. Applied to mammalian brain, this label-free cryogenic imaging method allows visualization of myelinated axons and sub-cellular features such as age-related pigmented cellular inclusions at a spatial resolution of similar to 100 nanometers and thicknesses approaching 100 microns. Because our approach does not require dehydration, staining or reduction of the sample, we introduce the possibility for subsequent analysis of the same tissue using orthogonal approaches that are expected to yield direct complementary insight to the biological features of interest.

Membrane Association Landscape of Myelin Basic Protein Portrays Formation of the Myelin Major Dense Line

A. Raasakka; S. Ruskamo; J. Kowal; R. Barker; A. Baumann et al. 

Compact myelin comprises most of the dry weight of myelin, and its insulative nature is the basis for saltatory conduction of nerve impulses. The major dense line (MDL) is a 3-nm compartment between two cytoplasmic leaflets of stacked myelin membranes, mostly occupied by a myelin basic protein (MBP) phase. MBP is an abundant myelin protein involved in demyelinating diseases, such as multiple sclerosis. The association of MBP with lipid membranes has been studied for decades, but the MBP-driven formation of the MDL remains elusive at the biomolecular level. We employed complementary biophysical methods, including atomic force microscopy, cryo-electron microscopy, and neutron scattering, to investigate the formation of membrane stacks all the way from MBP binding onto a single membrane leaflet to the organisation of a stable MDL. Our results support the formation of an amorphous protein phase of MBP between two membrane bilayers and provide a molecular model for MDL formation during myelination, which is of importance when understanding myelin assembly and demyelinating conditions.

Lipid Internal Dynamics Probed in Nanodiscs

D. Martinez; M. Decossas; J. Kowal; L. Frey; H. Stahlberg et al. 

Nanodiscs offer a very promising tool to incorporate membrane proteins into native-like lipid bilayers and an alternative to liposomes to maintain protein functions and protein-lipid interactions in a soluble nanoscale object. The activity of the incorporated membrane protein appears to be correlated to its dynamics in the lipid bilayer and by protein-lipid interactions. These two parameters depend on the lipid internal dynamics surrounded by the lipid-encircling discoidal scaffold protein that might differ from more unrestricted lipid bilayers observed in vesicles or cellular extracts. A solid-state NMR spectroscopy investigation of lipid internal dynamics and thermotropism in nanodiscs is reported. The gel-to-fluid phase transition is almost abolished for nanodiscs, which maintain lipid fluid properties for a large temperature range. The addition of cholesterol allows fine-tuning of the internal bilayer dynamics by increasing chain ordering. Increased site-specific order parameters along the acyl chain reflect a higher internal ordering in nanodiscs compared with liposomes at room temperature; this is induced by the scaffold protein, which restricts lipid diffusion in the nanodisc area.

Membrane vesicle secretion and prophage induction in multidrug-resistant Stenotrophomonas maltophilia in response to ciprofloxacin stress

S. Devos; W. Van Putte; J. Vitse; G. Van Driessche; S. Stremersch et al. 

Several bacterial species produce membrane vesicles (MVs) in response to antibiotic stress. However, the biogenesis and role of MVs in bacterial antibiotic resistance mechanisms have remained unclear. Here, we studied the effect of the fluoroquinolone ciprofloxacin on MV secretion by Stenotrophomonas maltophilia using a combination of electron microscopy and proteomic approaches. We found that in addition to the classical outer membrane vesicles (OMV), ciprofloxacin-stimulated cultures produced larger vesicles containing both outer and inner membranes termed outer-inner membrane vesicles (OIMV), and that such MVs are enriched with cytosolic proteins. Remarkably, OIMV were found to be decorated with filamentous structures identified as fimbriae. In addition, ciprofloxacin stress leads to the release of bacteriophages and phage tail-like particles. Prophage induction by ciprofloxacin has been linked to pathogenesis and horizontal gene transfer in several bacterial species. Together, our findings show that ciprofloxacin treatment of S. maltophilia leads to the secretion of a heterogeneous pool of MVs and the induction of prophages that are potentially involved in adverse side-effects during antibiotic treatment.

Direct protein crystallization on ultrathin membranes for diffraction measurements at X-ray free-electron lasers

N. Opara; I. Martiel; S. A. Arnold; T. Braun; H. Stahlberg et al. 

A new era of protein crystallography started when X-ray free-electron lasers (XFELs) came into operation, as these provide an intense source of X-rays that facilitates data collection in the ‘iffract-before-destroy’ regime. In typical experiments, crystals sequentially delivered to the beam are exposed to X-rays and destroyed. Therefore, the novel approach of serial crystallography requires thousands of nearly identical samples. Currently applied sample-delivery methods, in particular liquid jets or drop-on-demand systems, suffer from significant sample consumption of the precious crystalline material. Direct protein microcrystal growth by the vapour diffusion technique inside arrays of nanolitre-sized wells is a method specifically tailored to crystallography at XFELs. The wells, with X-ray transparent Si3N4 windows as bottoms, are fabricated in silicon chips. Their reduced dimensions can significantly decrease protein specimen consumption. Arrays provide crystalline samples positioned in an ordered way without the need to handle fragile crystals. The nucleation process inside these microfabricated cavities was optimized to provide high membrane coverage and a quasi-random crystal distribution. Tight sealing of the chips and protection of the crystals from dehydration were achieved, as confirmed by diffraction experiments at a protein crystallography beamline. Finally, the test samples were shown to be suitable for time-resolved measurements at an XFEL at femtosecond resolution.

Structure of the human multidrug transporter ABCG2

N. M. I. .. Taylor; I. Manolaridis; S. M. Jackson; J. Kowal; H. Stahlberg et al. 

ABCG2 is a constitutively expressed ATP-binding cassette (ABC) transporter that protects many tissues against xenobiotic molecules. Its activity affects the pharmacokinetics of commonly used drugs and limits the delivery of therapeutics into tumour cells, thus contributing to multidrug resistance. Here we present the structure of human ABCG2 determined by cryo-electron microscopy, providing the first high-resolution insight into a human multidrug transporter. We visualize ABCG2 in complex with two antigen-binding fragments of the human-specific, inhibitory antibody 5D3 that recognizes extracellular loops of the transporter. We observe two cholesterol molecules bound in the multidrug-binding pocket that is located in a central, hydrophobic, inward-facing translocation pathway between the transmembrane domains. Combined with functional in vitro analyses, our results suggest a multidrug recognition and transport mechanism of ABCG2, rationalize disease-causing single nucleotide polymorphisms and the allosteric inhibition by the 5D3 antibody, and provide the structural basis of cholesterol recognition by other G-subfamily ABC transporters.

Focus: The interface between data collection and data processing in cryo-EM

N. Biyani; R. D. Righetto; R. McLeod; D. Caujolle-Bert; D. Castano-Diez et al. 

We present a new software package called Focus that interfaces cryo-transmission electron microscopy (cryo-EM) data collection with computer image processing. Focus creates a user-friendly environment to import and manage data recorded by direct electron detectors and perform elemental image processing tasks in a high-throughput manner while new data is being acquired at the microscope. It provides the functionality required to remotely monitor the progress of data collection and data processing, which is essential now that automation in cryo-EM allows a steady flow of images of single particles, two-dimensional crystals, or electron tomography data to be recorded in overnight sessions. The rapid detection of any errors that may occur greatly increases the productivity of recording sessions at the electron microscope. (C) 2017 The Author(s). Published by Elsevier Inc.

Robust image alignment for cryogenic transmission electron microscopy

R. A. McLeod; J. Kowal; P. Ringler; H. Stahlberg 

Cryo-electron microscopy recently experienced great improvements in structure resolution due to direct electron detectors with improved contrast and fast read-out leading to single electron counting. High frames rates enabled dose fractionation, where a long exposure is broken into a movie, permitting specimen drift to be registered and corrected. The typical approach for image registration, with high shot noise and low contrast, is multi-reference (MR) cross-correlation. Here we present the software package Zorro, which provides robust drift correction for dose fractionation by use of an intensity-normalized cross-correlation and logistic noise model to weight each cross-correlation in the MR model and filter each cross-correlation optimally. Frames are reliably registered by Zorro with low dose and defocus. Methods to evaluate performance are presented, by use of independently-evaluated even- and odd frame stacks by trajectory comparison and Fourier ring correlation. Alignment of tiled sub-frames is also introduced, and demonstrated on an example dataset. Zorro source code is available at github.com/CINA/zorro. (C) 2016 Elsevier Inc. All rights reserved.

Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts

S. A. Arnold; S. Albiez; A. Bieri; A. Syntychaki; R. Adaixo et al. 

We present a sample preparation method for cryo-electron microscopy (cryo-EM) that requires only 320 nL of sample to prepare a cryo-EM grid, depending on the protocol used. The sample is applied and spread on the grid by a microcapillary. The procedure does not involve any blotting steps, and realtime monitoring allows the water film thickness to be assessed and decreased to an optimum value prior to vitrification. We demonstrate that the method is suitable for high-resolution cryo-EM and will enable alternative electron microscopy approaches, such as single-cell visual proteomics. (C) 2016 The Author(s). Published by Elsevier Inc.

Amyloid Fibril Polymorphism: Almost Identical on the Atomic Level, Mesoscopically Very Different

C. Seuring; J. Verasdonck; P. Ringler; R. Cadalbert; H. Stahlberg et al. 

Amyloid polymorphism of twisted and straight beta-endorphin fibrils was studied by negative-stain transmission electron microscopy, scanning transmission electron microscopy, and solid-state nuclear magnetic resonance spectroscopy. Whereas fibrils assembled in the presence of salt formed flat, striated ribbons, in the absence of salt they formed mainly twisted filaments. To get insights into their structural differences at the atomic level, 3D solid-state NMR spectra of both fibril types were acquired, allowing the detection of the differences in chemical shifts of C-13 and N-15 atoms in both preparations. The spectral fingerprints and therefore the chemical shifts are very similar for both fibril types. This indicates that the monomer structure and the molecular interfaces are almost the same but that these small differences do propagate to produce flat and twisted morphologies at the mesoscopic scale. This finding is in agreement with both experimental and theoretical considerations on the assembly of polymers (including amyloids) under different salt conditions, which attribute the mesoscopic difference of flat versus twisted fibrils to electrostatic intermolecular repulsions.

Dynamo Catalogue: Geometrical tools and data management for particle picking in subtomogram averaging of cryo-electron tomograms

D. Castano-Diez; M. Kudryashev; H. Stahlberg 

Cryo electron tomography allows macromolecular complexes within vitrified, intact, thin cells or sections thereof to be visualized, and structural analysis to be performed in situ by averaging over multiple copies of the same molecules. Image processing for subtomogram averaging is specific and cumbersome, due to the large amount of data and its three dimensional nature and anisotropic resolution. Here, we streamline data processing for subtomogram averaging by introducing an archiving system, Dynamo Catalogue. This system manages tomographic data from multiple tomograms and allows visual feedback during all processing steps, including particle picking, extraction, alignment and classification. The file structure of a processing project file structure includes logfiles of performed operations, and can be backed up and shared between users. Command line commands, database queries and a set of GUIs give the user versatile control over the process. Here, we introduce a set of geometric tools that streamline particle picking from simple (filaments, spheres, tubes, vesicles) and complex geometries (arbitrary 2D surfaces, rare instances on proteins with geometric restrictions, and 2D and 3D crystals). Advanced functionality, such as manual alignment and subboxing, is useful when initial templates are generated for alignment and for project customization. Dynamo Catalogue is part of the open source package Dynamo and includes tools to ensure format compatibility with the subtomogram averaging functionalities of other packages, such as jsubtomo, PyTom, PEET, EMAN2, XMIPP and Relion. (C) 2016 Published by Elsevier Inc.

Monitoring the Conformational Changes of Individual Cyclic Nucleotide-Gated Ion Channels by High-Speed Atomic Force Microscopy

M. Rangl; A. Miyagi; J. Kowal; H. Stahlberg; C. M. Nimigean et al. 

Proteoliposomes – a system to study membrane proteins under buffer gradients by cryo-EM

K. Sejwal; M. Chami; P. Baumgartner; J. Kowal; S. A. Mueller et al. 

Membrane proteins are vital to life and major therapeutic targets. Yet, understanding how they function is limited by a lack of structural information. In biological cells, membrane proteins reside in lipidic membranes and typically experience different buffer conditions on both sides of the membrane or even electric potentials and transmembrane gradients across the membranes. Proteoliposomes, which are lipidic vesicles filled with reconstituted membrane proteins, provide an ideal model system for structural and functional studies of membrane proteins under conditions that mimic nature to a certain degree. We discuss methods for the formation of liposomes and proteoliposomes, their imaging by cryo-electron microscopy, and the structural analysis of proteins present in their bilayer. We suggest the formation of ordered arrays akin to weakly ordered two-dimensional (2D) crystals in the bilayer of liposomes as a means to achieve high-resolution, and subsequent buffer modification as a method to capture snapshots of membrane proteins in action.

Expression, Biochemistry, and Stabilization with Camel Antibodies of Membrane Proteins: Case Study of the Mouse 5-HT3 Receptor

Cell-free reconstitution reveals centriole cartwheel assembly mechanisms

P. Guichard; V. Hamel; M. Le Guennec; N. Banterle; I. Iacovache et al. 

How cellular organelles assemble is a fundamental question in biology. The centriole organelle organizes around a nine-foldsymmetrical cartwheel structure typically 100 nm high comprising a stack of rings that each accommodates nine homodimers of SAS-6 proteins. Whether nine- fold symmetrical ring-like assemblies of SAS-6 proteins harbour more peripheral cartwheel elements is unclear. Furthermore, the mechanisms governing ring stacking are not known. Here we develop a cell-free reconstitution system for core cartwheel assembly. Using cryo-electron tomography, we uncover that the Chlamydomonas reinhardtii proteins CrSAS-6 and Bld10p together drive assembly of the core cartwheel. Moreover, we discover that CrSAS-6 possesses autonomous properties that ensure self-organized ring stacking. Mathematical fitting of reconstituted cartwheel height distribution suggests a mechanism whereby preferential addition of pairs of SAS- 6 rings governs cartwheel growth. In conclusion, we have developed a cell-free reconstitution system that reveals fundamental assembly principles at the root of centriole biogenesis.

Two-dimensional crystallization of the mouse serotonin 5-HT3A receptor

J. Rheinberger; G. Hassaine; M. Chami; H. Stahlberg; H. Vogel et al. 

The mouse serotonin 5-HT3A receptor is a homo-pentameric ligand-gated ion channel (pLGIC) mediating fast excitatory neurotransmission in the central nervous system. The molecular mechanism of ion permeation of 5-HT3A receptors triggered by the neurotransmitter serotonin is not yet fully understood. The recent X-ray structure of the mouse serotonin 5-HT3A receptor in complex with a stabilizing nanobody revealed for the first time the entire structure of a mammalian pLGIC in detergent. Structural information of the receptor in a lipid bilayer however is still limited primarily due to the lack of 2D crystals of the receptor in a lipid bilayer. Here we present our results on the formation and improvement of diffracting 2D crystals of the mouse 5-HT3A by limited proteolysis and addition of conformational nanobodies. (C) 2016 Elsevier Ltd. All rights reserved.

Solution structure of discoidal high-density lipoprotein particles with a shortened apolipoprotein A-I

S. Bibow; Y. Polyhach; C. Eichmann; C. N. Chi; J. Kowal et al. 

High-density lipoprotein (HDL) particles are cholesterol and lipid transport containers. Mature HDL particles destined for the liver develop through the formation of intermediate discoidal HDL particles, which are the primary acceptors for cholesterol. Here we present the three-dimensional structure of reconstituted discoidal HDL (rdHDL) particles, using a shortened construct of human apolipoprotein A-I, determined from a combination of nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR) and transmission electron microscopy (TEM) data. The rdHDL particles feature a protein double belt surrounding a lipid bilayer patch in an antiparallel fashion. The integrity of this structure is maintained by up to 28 salt bridges and a zipper-like pattern of cation-pi interactions between helices 4 and 6. To accommodate a hydrophobic interior, a gross ‘right-to-right’ rotation of the helices after lipidation is necessary. The structure reflects the complexity required for a shuttling container to hold a fluid lipid or cholesterol interior at a protein: lipid ratio of 1:50.

Real-time visualization of conformational changes within single MloK1 cyclic nucleotide-modulated channels

M. Rang; A. Miyagi; J. Kowal; H. Stahlberg; C. M. Nimigean et al. 

Eukaryotic cyclic nucleotide-modulated (CNM) ion channels perform various physiological roles by opening in response to cyclic nucleotides binding to a specialized cyclic nucleotide-binding domain. Despite progress in structure-function analysis, the conformational rearrangements underlying the gating of these channels are still unknown. Here, we image ligand-induced conformational changes in single CNM channels from Mesorhizobium loti (MloK1) in real-time, using high-speed atomic force microscopy. In the presence of cAMP, most channels are in a stable conformation, but a few molecules dynamically switch back and forth (blink) between at least two conformations with different heights. Upon cAMP depletion, more channels start blinking, with blinking heights increasing over time, suggestive of slow, progressive loss of ligands from the tetramer. We propose that during gating, MloK1 transitions from a set of mobile conformations in the absence to a stable conformation in the presence of ligand and that these conformations are central for gating the pore.

Biochemical and biophysical approaches to study the structure and function of the chloride channel (ClC) family of proteins

P. D. Abeyrathne; M. Chami; H. Stahlberg 

The chloride channel (ClC) protein family comprises both chloride (Cl-) channels and chloride/proton (Cl/H+) antiporters. In prokaryotes and eukaryotes, these proteins mediate the movement of Cl- ions across the membrane. In eukaryotes, ClC proteins play a role in the stabilization of membrane potential, epithelial ion transport, hippocampal neuroprotection, cardiac pacemaker activity and vesicular acidification. Moreover, mutations in the genes encoding ClC proteins can cause genetic disease in humans. In prokaryotes, the Cl/H+ antiporters, such as ClC-ec1 found in Escherichia coli promote proton expulsion in the extreme acid-resistance response common to enteric bacteria. To date, structural and functional studies of the prokaryotic protein have revealed unique structural features, including complicated transmembrane topology with 18 alpha-helices in each subunit and an anion-coordinating region in each subunit. Several different approaches such as X-ray crystallography, NMR, biochemical studies, and molecular dynamics simulations have been applied to the study of CIC proteins. Continued study of the unique structure and function of this diverse family of proteins has the potential to lead to the development of novel therapeutic targets for neuronal, renal, bone, and food-borne diseases. (C) 2016 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

Ultrathin membrane chips as X-ray transparent supports for serial crystallography

N. Opara; S. Arnold; T. Braun; H. Stahlberg; C. Padeste 

GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death

L. Sborgi; S. Ruhl; E. Mulvihill; J. Pipercevic; R. Heilig et al. 

Pyroptosis is a lytic type of cell death that is initiated by inflammatory caspases. These caspases are activated within multi-protein inflammasome complexes that assemble in response to pathogens and endogenous danger signals. Pyroptotic cell death has been proposed to proceed via the formation of a plasma membrane pore, but the underlying molecular mechanism has remained unclear. Recently, gasdermin D (GSDMD), a member of the ill-characterized gasdermin protein family, was identified as a caspase substrate and an essential mediator of pyroptosis. GSDMD is thus a candidate for pyroptotic pore formation. Here, we characterize GSDMD function in live cells and in vitro. We show that the N-terminal fragment of caspase-1-cleaved GSDMD rapidly targets the membrane fraction of macrophages and that it induces the formation of a plasma membrane pore. In vitro, the N-terminal fragment of caspase-1-cleaved recombinant GSDMD tightly binds liposomes and forms large permeability pores. Visualization of liposomeinserted GSDMD at nanometer resolution by cryo-electron and atomic force microscopy shows circular pores with variable ring diameters around 20 nm. Overall, these data demonstrate that GSDMD is the direct and final executor of pyroptotic cell death.

Total Sample Conditioning and Preparation of Nanoliter Volumes for Electron Microscopy

S. A. Arnold; S. Albiez; N. Opara; M. Chami; C. Schmidli et al. 

Electron microscopy (EM) entered a new era with the emergence of direct electron detectors and new nanocrystal electron diffraction methods. However, sample preparation techniques have not progressed and still suffer from extensive blotting steps leading to a massive loss of sample. Here, we present a simple but versatile method for the almost lossless sample conditioning and preparation of nanoliter volumes of biological samples for EM, keeping the sample under close to physiological condition. A microcapillary is used to aspirate 3-5 nL of sample. The microcapillary tip is immersed into a reservoir of negative stain or trehalose, where the sample becomes conditioned by diffusive exchange of salt and heavy metal ions or sugar molecules, respectively, before it is deposited as a small spot onto an EM grid. We demonstrate the use of the method to prepare protein particles for imaging by transmission EM and nanocrystals for analysis by electron diffraction. Furthermore, the minute sample volume required for this method enables alternative strategies for biological experiments, such as the analysis of the content of a single cell by visual proteomics, fully exploiting the single molecule detection limit of EM.

Cholesteryl ester transfer between lipoproteins does not require a ternary tunnel complex with CETP

M. E. Lauer; A. Graff-Meyer; A. C. Rufer; C. Maugeais; E. von der Mark et al. 

The cholesteryl ester transfer protein (CETP) enables the transfer of cholesteryl ester (CE) from high density lipoproteins (HDL) to low-density lipoproteins (LDL) in the plasma compartment. CETP inhibition raises plasma levels of HDL cholesterol; a ternary tunnel complex with CETP bridging HDL and LDL was suggested as a mechanism. Here, we test whether the inhibition of CETP tunnel complex formation is a promising approach to suppress CE transfer from HDL to LDL, for potential treatment of cardio-vascular disease (CVD). Three monoclonal antibodies against different epitopes of CETP are assayed for their potential to interfere with CE transfer between HDL and/or LDL. Surprisingly, antibodies that target the tips of the elongated CETP molecule, interaction sites sterically required to form the suggested transfer complexes, do not interfere with CETP activity, but an antibody binding to the central region does. We show that CETP interacts with HDL, but not with LDL. Our findings demonstrate that a ternary tunnel complex is not the mechanistic prerequisite to transfer CE among lipoproteins. (C) 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license.

Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs

F. Plath; P. Ringler; A. Graff-Meyer; H. Stahlberg; M. E. Lauer et al. 

The formation of undesired high molecular weight species such as dimers is an important quality attribute for therapeutic monoclonal antibody formulations. Therefore, the thorough understanding of mAb dimerization and the detailed characterization mAb dimers is of great interest for future pharmaceutical development of therapeutic antibodies. In this work, we focused on the analyses of different mAb dimers regarding size, surface properties, chemical identity, overall structure and localization of possible dimerization sites. Dimer fractions of different mAbs were isolated to a satisfactory purity from bulk material and revealed 2 predominant overall structures, namely elongated and compact dimer forms. The elongated dimers displayed one dimerization site involving the tip of the Fab domain. Depending on the stress applied, these elongated dimers are connected either covalently or non-covalently. In contrast, the compact dimers exhibited non-covalent association. Several interaction points were detected for the compact dimers involving the hinge region or the base of the Fab domain. These results indicate that mAb dimer fractions are rather complex and may contain more than one kind of dimer. Nevertheless, the overall appearance of mAb dimers suggests the existence of 2 predominant dimeric structures, elongated and compact, which are commonly present in preparations of therapeutic mAbs.

Cullin-RING ubiquitin E3 ligase regulation by the COP9 signalosome

S. Cavadini; E. S. Fischer; R. D. Bunker; A. Potenza; G. M. Lingaraju et al. 

The cullin-RING ubiquitin E3 ligase (CRL) family comprises over 200 members in humans. The COP9 signalosome complex (CSN) regulates CRLs by removing their ubiquitin-like activator NEDD8. The CUL4A-RBX1-DDB1-DDB2 complex (CRL4A(DDB2)) monitors the genome for ultraviolet-light-induced DNA damage. CRL4A(DBB2) is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4A(DDB2) and the principles of CSN activation are poorly understood. Here we present cryo-electron microscopy structures for CSN in complex with neddylated CRL4A ligases to 6.4 angstrom resolution. The CSN conformers defined by cryo-electron microscopy and a novel apo-CSN crystal structure indicate an induced-fit mechanism that drives CSN activation by neddylated CRLs. We find that CSN and a substrate cannot bind simultaneously to CRL4A, favouring a deneddylated, inactive state for substrate-free CRL4 complexes. These architectural and regulatory principles appear conserved across CRL families, allowing global regulation by CSN.

Preparation and Characterization of Stable -Synuclein Lipoprotein Particles

C. Eichmann; S. Campioni; J. Kowal; I. Maslennikov; J. Gerez et al. 

Multiple neurodegenerative diseases are caused by the aggregation of the human -Synuclein (-Syn) protein. -Syn possesses high structural plasticity and the capability of interacting with membranes. Both features are not only essential for its physiological function but also play a role in the aggregation process. Recently it has been proposed that -Syn is able to form lipid-protein particles reminiscent of high-density lipoproteins. Here, we present a method to obtain a stable and homogeneous population of nanometer-sized particles composed of -Syn and anionic phospholipids. These particles are called -Syn lipoprotein (nano)particles to indicate their relationship to high-density lipoproteins formed by human apolipoproteins in vivo and of in vitro self-assembling phospholipid bilayer nanodiscs. Structural investigations of the -Syn lipoprotein particles by circular dichroism (CD) and magic angle solid-state nuclear magnetic resonance (MAS SS-NMR) spectroscopy establish that -Syn adopts a helical secondary structure within these particles. Based on cryo-electron microscopy (cryo-EM) and dynamic light scattering (DLS) -Syn lipoprotein particles have a defined size with a diameter of approximate to 23 nm. Chemical cross-linking in combination with solution-state NMR and multiangle static light scattering (MALS) of -Syn particles reveal a high-order protein-lipid entity composed of approximate to 8-10 -Syn molecules. The close resemblance in size between cross-linked in vitro-derived -Syn lipoprotein particles and a cross-linked species of endogenous -Syn from SH-SY5Y human neuroblastoma cells indicates a potential functional relevance of -Syn lipoprotein nanoparticles.

Direct Visualization of Glutamate Transporter Transport Cycle

Y. Ruan; A. Miyagi; X. Wang; M. Chami; H. Stahlberg et al. 

The lipidome associated with the gamma-secretase complex is required for its integrity and activity

S. Ayciriex; H. Gerber; G. M. G. Osuna; M. Chami; H. Stahlberg et al. 

gamma-Secretase is a multi-subunit membrane protease complex that catalyses the final intramembrane cleavage of the beta-amyloid precursor protein (APP) during the neuronal production of amyloid-beta peptides (A beta), which are implicated as the causative agents of Alzheimer’s disease (AD). In the present study, we report the reconstitution of a highly purified, active gamma-secretase complex into proteoliposomes without exogenous lipids and provide the first direct evidence for the existence of a microenvironment of 53 molecular species from 11 major lipid classes specifically associated with the gamma-secretase complex, including phosphatidylcholine and cholesterol. Importantly, we demonstrate that the pharmacological modulation of certain phospholipids abolishes both the integrity and the enzymatic activity of the intramembrane protease. Together, our findings highlight the importance of a specific lipid microenvironment for the structure and function of gamma-secretase.

Structure of the T4 baseplate and its function in triggering sheath contraction

N. M. I. Taylor; N. S. Prokhorov; R. C. Guerrero-Ferreira; M. M. Shneider; C. Browning et al. 

Several systems, including contractile tail bacteriophages, the type VI secretion system and R-type pyocins, use a multiprotein tubular apparatus to attach to and penetrate host cell membranes. This macromolecular machine resembles a stretched, coiled spring (or sheath) wound around a rigid tube with a spike-shaped protein at its tip. A baseplate structure, which is arguably the most complex part of this assembly, relays the contraction signal to the sheath. Here we present the atomic structure of the approximately 6-megadalton bacteriophage T4 baseplate in its pre- and post-host attachment states and explain the events that lead to sheath contraction in atomic detail. We establish the identity and function of a minimal set of components that is conserved in all contractile injection systems and show that the triggering mechanism is universally conserved.

The Structure of the Mouse Serotonin 5-HT3 Receptor in Lipid Vesicles

M. Kudryashev; D. Castaño-Díez; C. Deluz; G. Hassaine; L. Grasso et al. 

The function of membrane proteins is best understood if their structure in the lipid membrane is known. Here, we determined the structure of the mouse serotonin 5-HT3 receptor inserted in lipid bilayers to a resolution of 12 Å without stabilizing antibodies by cryo electron tomography and subtomogram averaging. The reconstruction reveals protein secondary structure elements in the transmembrane region, the extracellular pore, and the transmembrane channel pathway, showing an overall similarity to the available X-ray model of the truncated 5-HT3 receptor determined in the presence of a stabilizing nanobody. Structural analysis of the 5-HT3 receptor embedded in a lipid bilayer allowed the position of the membrane to be determined. Interactions between the densely packed receptors in lipids were visualized, revealing that the interactions were maintained by the short horizontal helices. In combination with methodological improvements, our approach enables the structural analysis of membrane proteins in response to voltage and ligand gating.

Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy

L. Sborgi; F. Ravotti; V. P. Dandey; M. S. Dick; A. Mazur et al. 

Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous triggers. Assembly of inflammasomes is induced by activation of a receptor protein. Many inflammasome receptors require the adapter protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)], which consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1 recruitment. Here, we characterize the structure and filament formation of mouse ASC in vitro at atomic resolution. Information from cryo-electron microscopy and solid-state NMR spectroscopy is combined in a single structure calculation to obtain the atomic-resolution structure of the ASC filament. Perturbations of NMR resonances upon filament formation monitor the specific binding interfaces of ASC-PYD association. Importantly, NMR experiments show the rigidity of the PYD forming the core of the filament as well as the high mobility of the CARD relative to this core. The findings are validated by structure-based mutagenesis experiments in cultured macrophages. The 3D structure of the mouse ASC-PYD filament is highly similar to the recently determined human ASC-PYD filament, suggesting evolutionary conservation of ASC-dependent inflammasome mechanisms.

Translational arrest by a prokaryotic signal recognition particle is mediated by RNA interactions

B. Beckert; A. Kedrov; D. Sohmen; G. Kempf; K. Wild et al. 

The signal recognition particle (SRP) recognizes signal sequences of nascent polypeptides and targets ribosome-nascent chain complexes to membrane translocation sites. In eukaryotes, translating ribosomes are slowed down by the Alu domain of SRP to allow efficient targeting. In prokaryotes, however, little is known about the structure and function of Alu domain-containing SRPs. Here, we report a complete molecular model of SRP from the Gram-positive bacterium Bacillus subtilis, based on cryo-EM. The SRP comprises two subunits, 6S RNA and SRP54 or Ffh, and it facilitates elongation slowdown similarly to its eukaryotic counterpart. However, protein contacts with the small ribosomal subunit observed for the mammalian Alu domain are substituted in bacteria by RNA-RNA interactions of 6S RNA with the a-sarcin-ricin loop and helices H43 and H44 of 23S rRNA. Our findings provide a structural basis for cotranslational targeting and RNA-driven elongation arrest in prokaryotes.

3D reconstruction of two-dimensional crystals

H. Stahlberg; N. Biyani; A. Engel 

Electron crystallography of two-dimensional (2D) crystals determines the structure of membrane proteins in the lipid bilayer by imaging with cryo-electron microscopy and image processing. Membrane proteins can be packed in regular 2D arrays by their reconstitution in the presence of lipids at low lipid to protein weight-to-weight ratio. The crystal quality depends on the protein purity and homogeneity, its stability, and on the crystallization conditions. A 2D crystal presents the membrane protein in a functional and fully lipidated state. Electron crystallography determines the 3D structure even of small membrane proteins up to atomic resolution, but 3D density maps have a better resolution in the membrane plane than in the vertical direction. This problem can be partly eliminated by applying an iterative algorithm that exploits additional known constraints about the 2D crystal. 2D electron crystallography is particularly attractive for the structural analysis of membrane proteins that are too small for single particle analyses and too unstable to form 3D crystals. With the recent introduction of direct electron detector cameras, the routine determination of the atomic 3D structure of membrane-embedded membrane proteins is in reach. (C) 2015 Published by Elsevier Inc.

CTF Challenge: Result summary

R. Marabini; B. Carragher; S. Chen; J. Chen; A. Cheng et al. 

Image formation in bright field electron microscopy can be described with the help of the contrast transfer function (CTF). In this work the authors describe the “CTF Estimation Challenge”, called by the Madrid Instruct Image Processing Center (I2PC) in collaboration with the National Center for Macromolecular Imaging (NCMI) at Houston. Correcting for the effects of the CTF requires accurate knowledge of the CTF parameters, but these have often been difficult to determine. In this challenge, researchers have had the opportunity to test their ability in estimating some of the key parameters of the electron microscope CTF on a large micrograph data set produced by well-known laboratories on a wide set of experimental conditions. This work presents the first analysis of the results of the CTF Estimation Challenge, including an assessment of the performance of the different software packages under different conditions, so as to identify those areas of research where further developments would be desirable in order to achieve high-resolution structural information. (C) 2015 Elsevier Inc. All rights reserved.

Structure of the Type VI Secretion System Contractile Sheath

M. Kudryashev; R. Y-R. Wang; M. Brackmann; S. Scherer; T. Maier et al. 

Bacteria use rapid contraction of a long sheath of the type VI secretion system (T6SS) to deliver effectors into a target cell. Here, we present an atomic-resolution structure of a native contracted Vibrio cholerae sheath determined by cryo-electron microscopy. The sheath subunits, composed of tightly interacting proteins VipA and VipB, assemble into a six-start helix. The helix is stabilized by a core domain assembled from four b strands donated by one VipA and two VipB molecules. The fold of inner and middle layers is conserved between T6SS and phage sheaths. However, the structure of the outer layer is distinct and suggests a mechanism of interaction of the bacterial sheath with an accessory ATPase, ClpV, that facilitates multiple rounds of effector delivery. Our results provide a mechanistic insight into assembly of contractile nanomachines that bacteria and phages use to translocate macromolecules across membranes.

Yersinia enterocolitica type III secretion injectisomes form regularly spaced clusters, which incorporate new machines upon activation

M. Kudryashev; A. Diepold; M. Amstutz; J. P. Armitage; H. Stahlberg et al. 

Bacterial type III secretion systems or injectisomes are multiprotein complexes directly transporting bacterial effector proteins into eukaryotic host cells. To investigate the distribution of injectisomes in the bacterium and the influence of activation of the system on that distribution, we combined in vivo fluorescent imaging and high-resolution in situ visualization of Yersinia enterocolitica injectisomes by cryo-electron tomography. Fluorescence microscopy showed the injectisomes as regularly distributed spots around the bacterial cell. Under secreting conditions (absence of Ca2+), the intensity of single spots significantly increased compared with non-secreting conditions (presence of Ca2+), in line with an overall up-regulation of expression levels of all components. Single injectisomes observed by cryo-electron tomography tended to cluster at distances less than 100nm, suggesting that the observed fluorescent spots correspond to evenly distributed clusters of injectisomes, rather than single injectisomes. The up-regulation of injectisome components led to an increase in the number of injectisomes per cluster rather than the formation of new clusters. We suggest that injectisome clustering may allow more effective secretion into the host cells.

Disease Modeling and Phenotypic Drug Screening for Diabetic Cardiomyopathy using Human Induced Pluripotent Stem Cells

F. M. Drawnel; S. Boccardo; M. Prummer; F. Delobel; A. Graff et al. 

Diabetic cardiomyopathy is a complication of type 2 diabetes, with known contributions of lifestyle and genetics. We develop environmentally and genetically driven in vitro models of the condition using human-induced-pluripotent-stem-cell-derived cardiomyocytes. First, we mimic diabetic clinical chemistry to induce a phenotypic surrogate of diabetic cardiomyopathy, observing structural and functional disarray. Next, we consider genetic effects by deriving cardiomyocytes from two diabetic patients with variable disease progression. The cardiomyopathic phenotype is recapitulated in the patient-specific cells basally, with a severity dependent on their original clinical status. These models are incorporated into successive levels of a screening platform, identifying drugs that preserve cardiomyocyte phenotype in vitro during diabetic stress. In this work, we present a patient-specific induced pluripotent stem cell (iPSC) model of a complex metabolic condition, showing the power of this technique for discovery and testing of therapeutic strategies for a disease with ever-increasing clinical significance.

The ultrastructure of Chlorobaculum tepidum revealed by cryo-electron tomography

M. Kudryashev; A. Aktoudianaki; D. Dedoglou; H. Stahlberg; G. Tsiotis 

Chlorobaculum (Cba) tepidum is a green sulfur bacterium that oxidizes sulfide, elemental sulfur, and thiosulfate for photosynthetic growth. As other anoxygenic green photosynthetic bacteria, Cba tepidum synthesizes bacteriochlorophylls for the assembly of a large light-harvesting antenna structure, the chlorosome. Chlorosomes are sac-like structures that are connected to the reaction centers in the cytoplasmic membrane through the BChl alpha-containing Fenna-Matthews-Olson protein. Most components of the photosynthetic machinery are known on a biophysical level, however, the structural integration of light harvesting with charge separation is still not fully understood. Despite over two decades of research, gaps in our understanding of cellular architecture exist. Here we present an in-depth analysis of the cellular architecture of the thermophilic photosynthetic green sulfur bacterium of Cba tepidum by cryo-electron tomography. We examined whole hydrated cells grown under different electron donor conditions. Our results reveal the distribution of chlorosomes in 3D in an unperturbed cell, connecting elements between chlorosomes and the cytoplasmic membrane and the distribution of reaction centers in the cytoplasmic membrane. (C) 2014 Elsevier B.V. All rights reserved.

Functional surface engineering by nucleotide-modulated potassium channel insertion into polymer membranes attached to solid supports

J. L. Kowal; J. K. Kowal; D. Wu; H. Stahlberg; C. G. Palivan et al. 

Planar solid-supported membranes based on amphiphilic block copolymers represent promising systems for the artificial creation of structural surfaces. Here we introduce a method for engineering functional planar solid-supported membranes through insertion of active biomolecules. We show that membranes based on poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PDMS-b-PMOXA) amphiphilic diblock copolymers, which mimic natural membranes, are suitable for hosting biomolecules. Our strategy allows preparation of large-area, well-ordered polymer bilayers via Langmuir-Blodgett and Langmuir-Schaefer transfers, and insertion of biomolecules by using Bio-Beads. We demonstrate that a model membrane protein, the potassium channel from the bacterium Mesorhizobium loti, remains functional after insertion into the planar solid-supported polymer membrane. This approach can be easily extended to generate a platform of functional solid-supported membranes by insertion of different hydrophobic biomolecules, and employing different types of solid substrates for desired applications. (C) 2014 Elsevier Ltd. All rights reserved.

Structural and Mechanistic Paradigm of Leptin Receptor Activation Revealed by Complexes with Wild-Type and Antagonist Leptins

K. Moharana; L. Zabeau; F. Peelman; P. Ringler; H. Stahlberg et al. 

Leptin activates its cognate receptor (LR) to regulate body weight and metabolically costly processes, such as reproduction and immune responses. Despite such benevolent pleiotropy, leptin-mediated signaling has been implicated in autoimmune diseases and breast cancer, thereby rejuvenating interest in leptin antagonism. We present comparative biochemical and structural studies of the LR ectodomain (LRecto) in complex with wild-type and antagonist leptin variants. We show that high-affinity binding of leptin to the cytokine receptor homology 2 domain of LRecto primes interactions with the Ig-domain (LRIg) of another leptin-bound LRecto to establish a quaternary assembly. In contrast, antagonist leptin variants carrying mutations at the LRIg binding site only enable binary complexes with LRecto. Acetylation of free cysteines in LRecto also abrogates quaternary complexes, suggesting a functional role for intrareceptor disulfides. We propose a revised conceptual framework for LR activation whereby leptin activates predimerized LR at the cell surface to seed higher order complexes with 4:4 stoichiometry.

Exploring the Interactome: Microfluidic Isolation of Proteins and Interacting Partners for Quantitative Analysis by Electron Microscopy

D. Giss; S. Kemmerling; V. Dandey; H. Stahlberg; T. Braun 

Multimolecular protein complexes are important for many cellular processes. However, the stochastic nature of the cellular interactome makes the experimental detection of complex protein assemblies difficult and quantitative analysis at the single molecule level essential. Here, we present a fast and simple microfluidic method for (i) the quantitative isolation of endogenous levels of untagged protein complexes from minute volumes of cell lysates under close to physiological conditions and (ii) the labeling of specific components constituting these complexes. The method presented uses specific antibodies that are conjugated via a photocleavable linker to magnetic beads that are trapped in microcapillaries to immobilize the target proteins. Proteins are released by photocleavage, eluted, and subsequently analyzed by quantitative transmission electron microscopy at the single molecule level. Additionally, before photocleavage, immunogold can be employed to label proteins that interact with the primary target protein. Thus, the presented method provides a new way to study the interactome and, in combination with single molecule transmission electron microscopy, to structurally characterize the large, dynamic, heterogeneous multimolecular protein complexes formed.

2dx_automator: Implementation of a semiautomatic high-throughput high-resolution cryo-electron crystallography pipeline

S. Scherer; J. Kowal; M. Chami; V. Dandey; M. Arheit et al. 

The introduction of direct electron detectors (DED) to cryo-electron microscopy has tremendously increased the signal-to-noise ratio (SNR) and quality of the recorded images. We discuss the optimal use of DEDs for cryo-electron crystallography, introduce a new automatic image processing pipeline, and demonstrate the vast improvement in the resolution achieved by the use of both together, especially for highly tilted samples. The new processing pipeline (now included in the software package 2dx) exploits the high SNR and frame readout frequency of DEDs to automatically correct for beam-induced sample movement, and reliably processes individual crystal images without human interaction as data are being acquired. A new graphical user interface (GUI) condenses all information required for quality assessment in one window, allowing the imaging conditions to be verified and adjusted during the data collection session. With this new pipeline an automatically generated unit cell projection map of each recorded 2D crystal is available less than 5 min after the image was recorded. The entire processing procedure yielded a three-dimensional reconstruction of the 2D-crystallized ion-channel membrane protein Ml0K1 with a much-improved resolution of 5 angstrom in-plane and 7 angstrom in the z-direction, within 2 days of data acquisition and simultaneous processing. The results obtained are superior to those delivered by conventional photographic film-based methodology of the same sample, and demonstrate the importance of drift-correction. (c) 2014 The Authors. Published by Elsevier Inc.

Relative bioavailability of a new tamper resistant extended-release oxycodone/naloxone combination product

H. Stahlberg; M. Brett; S. Schwier; A. Philipp 

Rendering graphene supports hydrophilic with non-covalent aromatic functionalization for transmission electron microscopy

R. S. Pantelic; W. Fu; C. Schoenenberger; H. Stahlberg 

Amorphous carbon films have been routinely used to enhance the preparation of frozen-hydrated samples for transmission electron microscopy (TEM), either in retaining protein concentration, providing mechanical stability or dissipating sample charge. However, strong background signal from the amorphous carbon support obstructs that of the sample, and the insulating properties of thin amorphous carbon films preclude any efficiency in dispersing charge. Graphene addresses the limitations of amorphous carbon. Graphene is a crystalline material with virtually no phase or amplitude contrast and unparalleled, high electrical carrier mobility. However, the hydrophobic properties of graphene have prevented its routine application in Cryo-TEM. This Letter reports a method for rendering graphene TEM supports hydrophilic-a convenient approach maintaining graphene’s structural and electrical properties based on non-covalent, aromatic functionalization. (C) 2014 AIP Publishing LLC.

Single particle 3D reconstruction for 2D crystal images of membrane proteins

S. Scherer; M. Arheit; J. Kowal; X. Zeng; H. Stahlberg 

In cases where ultra-flat cryo-preparations of well-ordered two-dimensional (2D) crystals are available, electron crystallography is a powerful method for the determination of the high-resolution structures of membrane and soluble proteins. However, crystal unbending and Fourier-filtering methods in electron crystallography three-dimensional (3D) image processing are generally limited in their performance for 2D crystals that are badly ordered or non-flat. Here we present a single particle image processing approach, which is implemented as an extension of the 2D crystallographic pipeline realized in the 2dx software package, for the determination of high-resolution 3D structures of membrane proteins. The algorithm presented, addresses the low single-to-noise ratio (SNR) of 2D crystal images by exploiting neighborhood correlation between adjacent proteins in the 2D crystal. Compared with conventional single particle processing for randomly oriented particles, the computational costs are greatly reduced due to the crystal-induced limited search space, which allows a much finer search space compared to classical single particle processing. To reduce the considerable computational costs, our software features a hybrid parallelization scheme for multi-CPU clusters and computer with high-end graphic processing units (GPUs). We successfully apply the new refinement method to the structure of the potassium channel MloK1. The calculated 3D reconstruction shows more structural details and contains less noise than the map obtained by conventional Fourier-filtering based processing of the same 2D crystal images. (C) 2013 The Authors. Published by Elsevier Inc. All rights reserved.

A KcsA/MloK1 Chimeric Ion Channel Has Lipid-dependent Ligand-binding Energetics

J. G. McCoy; R. Rusinova; D. M. Kim; J. Kowal; S. Banerjee et al. 

Background: The mechanism of ligand gating in physiologically important cyclic nucleotide-modulated channels is unknown. Results: We constructed and purified a chimeric ion channel with activity modulated by cAMP and used it to measure ligand-binding energetics. Conclusion: cAMP binds with high lipid-dependent affinity to the chimeric channel. Significance: The availability of a good protein preparation enables assays that shed light on ligand gating.Cyclic nucleotide-modulated ion channels play crucial roles in signal transduction in eukaryotes. The molecular mechanism by which ligand binding leads to channel opening remains poorly understood, due in part to the lack of a robust method for preparing sufficient amounts of purified, stable protein required for structural and biochemical characterization. To overcome this limitation, we designed a stable, highly expressed chimeric ion channel consisting of the transmembrane domains of the well characterized potassium channel KcsA and the cyclic nucleotide-binding domains of the prokaryotic cyclic nucleotide-modulated channel MloK1. This chimera demonstrates KcsA-like pH-sensitive activity which is modulated by cAMP, reminiscent of the dual modulation in hyperpolarization-activated and cyclic nucleotide-gated channels that display voltage-dependent activity that is also modulated by cAMP. Using this chimeric construct, we were able to measure for the first time the binding thermodynamics of cAMP to an intact cyclic nucleotide-modulated ion channel using isothermal titration calorimetry. The energetics of ligand binding to channels reconstituted in lipid bilayers are substantially different from those observed in detergent micelles, suggesting that the conformation of the chimera’s transmembrane domain is sensitive to its (lipid or lipid-mimetic) environment and that ligand binding induces conformational changes in the transmembrane domain. Nevertheless, because cAMP on its own does not activate these chimeric channels, cAMP binding likely has a smaller energetic contribution to gating than proton binding suggesting that there is only a small difference in cAMP binding energy between the open and closed states of the channel.

Ligand-induced structural changes in the cyclic nucleotide-modulated potassium channel MloK1

J. Kowal; M. Chami; P. Baumgartner; M. Arheit; P-L. Chiu et al. 

Cyclic nucleotide-modulated ion channels are important for signal transduction and pacemaking in eukaryotes. The molecular determinants of ligand gating in these channels are still unknown, mainly because of a lack of direct structural information. Here we report ligand-induced conformational changes in full-length MloK1, a cyclic nucleotide-modulated potassium channel from the bacterium Mesorhizobium loti, analysed by electron crystallography and atomic force microscopy. Upon cAMP binding, the cyclic nucleotide-binding domains move vertically towards the membrane, and directly contact the S1-S4 voltage sensor domains. This is accompanied by a significant shift and tilt of the voltage sensor domain helices. In both states, the inner pore-lining helices are in an ‘open’ conformation. We propose a mechanism in which ligand binding can favour pore opening via a direct interaction between the cyclic nucleotide-binding domains and voltage sensors. This offers a simple mechanistic hypothesis for the coupling between ligand gating and voltage sensing in eukaryotic HCN channels.

Clostridium difficile toxin CDT hijacks microtubule organization and reroutes vesicle traffic to increase pathogen adherence

C. Schwan; A. S. Kruppke; T. Noelke; L. Schumacher; F. Koch-Nolte et al. 

Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis by the actions of Rho-glucosylating toxins A and B. Recently identified hypervirulent strains, which are associated with increased morbidity and mortality, additionally produce the actin-ADP-ribosylating toxin C. difficile transferase (CDT). CDT depolymerizes actin, causes formation of microtubule-based protrusions, and increases pathogen adherence. Here we show that CDT-induced protrusions allow vesicle traffic and contain endoplasmic reticulum tubules, connected to microtubules via the calcium sensor Stim1. The toxin reroutes Rab11-positive vesicles containing fibronectin, which is involved in bacterial adherence, from basolateral to the apical membrane sides in a microtubule-and Stim1-dependent manner. The data yield a model of C. difficile adherence regulated by actin depolymerization, microtubule restructuring, subsequent Stim1-dependent Ca2+ signaling, vesicle rerouting, and secretion of ECM proteins to increase bacterial adherence.

Expression, Purification and Stabilization of the Mouse 5HT3 Receptor

G. Hassaine; C. Deluz; A. Graff; C. Moreau; R. Wyss et al. 

X-ray structure of the mouse serotonin 5-HT3 receptor

G. Hassaine; C. Deluz; L. Grasso; R. Wyss; M. B. Tol et al. 

Neurotransmitter-gated ion channels of the Cys-loop receptor family mediate fast neurotransmission throughout the nervous system. The molecular processes of neurotransmitter binding, subsequent opening of the ion channel and ion permeation remain poorly understood. Here we present the X-ray structure of a mammalian Cys-loop receptor, the mouse serotonin 5-HT3 receptor, at 3.5 angstrom resolution. The structure of the proteolysed receptor, made up of two fragments and comprising part of the intracellular domain, was determined in complex with stabilizing nanobodies. The extracellular domain reveals the detailed anatomy of the neurotransmitter binding site capped by a nanobody. The membrane domain delimits an aqueous pore with a 4.6 angstrom constriction. In the intracellular domain, a bundle of five intracellular helices creates a closed vestibule where lateral portals are obstructed by loops. This 5-HT3 receptor structure, revealing part of the intracellular domain, expands the structural basis for understanding the operating mechanism of mammalian Cys-loop receptors.

openBEB: open biological experiment browser for correlative measurements

C. Ramakrishnan; A. Bieri; N. Sauter; S. Roizard; P. Ringler et al. 

Background: New experimental methods must be developed to study interaction networks in systems biology. To reduce biological noise, individual subjects, such as single cells, should be analyzed using high throughput approaches. The measurement of several correlative physical properties would further improve data consistency. Accordingly, a considerable quantity of data must be acquired, correlated, catalogued and stored in a database for subsequent analysis. Results: We have developed openBEB (open Biological Experiment Browser), a software framework for data acquisition, coordination, annotation and synchronization with database solutions such as openBIS. OpenBEB consists of two main parts: A core program and a plug-in manager. Whereas the data-type independent core of openBEB maintains a local container of raw-data and metadata and provides annotation and data management tools, all data-specific tasks are performed by plug-ins. The open architecture of openBEB enables the fast integration of plug-ins, e.g., for data acquisition or visualization. A macro-interpreter allows the automation and coordination of the different modules. An update and deployment mechanism keeps the core program, the plug-ins and the metadata definition files in sync with a central repository. Conclusions: The versatility, the simple deployment and update mechanism, and the scalability in terms of module integration offered by openBEB make this software interesting for a large scientific community. OpenBEB targets three types of researcher, ideally working closely together: (i) Engineers and scientists developing new methods and instruments, e.g., for systems-biology, (ii) scientists performing biological experiments, (iii) theoreticians and mathematicians analyzing data. The design of openBEB enables the rapid development of plug-ins, which will inherently benefit from the “house keeping” abilities of the core program. We report the use of openBEB to combine live cell microscopy, microfluidic control and visual proteomics. In this example, measurements from diverse complementary techniques are combined and correlated.

Cryo-electron microscopy of membrane proteins

Structure of the Dodecameric Yersinia enterocolitica Secretin YscC and Its Trypsin-Resistant Core

J. Kowal; M. Chami; P. Ringler; S. A. Mueller; M. Kudryashev et al. 

The type Ill secretion system machinery, also known as the injectisome, delivers bacterial effector proteins into eukaryotic cells during infection. The outer membrane YscC secretin is a major part of Yersinia enterocolitica’s injectisome and is among the first components to assemble, solely assisted by its pilotin, YscW. We have determined the three-dimensional structures of the native complex and its protease-resistant core to 12 A resolution by cryo-electron microscopy (cryo-EM) and show that YscC forms a dodecameric complex. Cryo-EM of YscC reconstituted into proteoliposomes defines the secretin’s membrane-spanning region. Native YscC consists of an outer membrane ring connected via a thin cylindrical wall to a conical, periplasmic region that exposes N-terminal petals connected by flexible linkers. These petals harbor the binding site of YscD, a component of the inner membrane ring. A change in their orientation adapts the length of the YscC secretin and facilitates its interaction with YscD.

Growth of Large and Highly Ordered 2D Crystals of a K + Channel, Structural Role of Lipidic Environment (vol 105, pg 398, 2013)

R. De Zorzi; W. V. Nicholson; J. M. Guigner; F. Erne-Brand; H. Stahlberg et al. 

Single-cell lysis for visual analysis by electron microscopy

S. Kemmerling; S. A. Arnold; B. A. Bircher; N. Sauter; C. Escobedo et al. 

The stochastic nature of biological systems makes the study of individual cells a necessity in systems biology. Yet, handling and disruption of single cells and the analysis of the relatively low concentrations of their protein components still challenges available techniques. Transmission electron microscopy (TEM) allows for the analysis of proteins at the single-molecule level. Here, we present a system for single-cell lysis under light microscopy observation, followed by rapid uptake of the cell lysate. Eukaryotic cells were grown on conductively coated glass slides and observed by light microscopy. A custom-designed microcapillary electrode was used to target and lyse individual cells with electrical pulses. Nanoliter volumes were subsequently aspirated into the microcapillary and dispensed onto an electron microscopy grid for TEM inspection. We show, that the cell lysis and preparation method conserves protein structures well and is suitable for visual analysis by TEM. (C) 2013 Elsevier Inc.. Published by Elsevier Inc. All rights reserved.

Vaccinia Virus Entry Is Followed by Core Activation and Proteasome-Mediated Release of the Immunomodulatory Effector VH1 from Lateral Bodies

F. I. Schmidt; C. K. E. Bleck; L. Reh; K. Novy; B. Wollscheid et al. 

Host cell entry of vaccinia virus, the prototypic poxvirus, involves a membrane fusion event delivering the viral core and two proteinaceous lateral bodies (LBs) into the cytosol. Uncoating of viral cores is poorly characterized, and the composition and function of LBs remains enigmatic. We found that cytosolic cores rapidly dissociated from LBs and expanded in volume, which coincided with reduction of disulfide-bonded core proteins. We identified the abundant phosphoprotein F17, the dual-specificity phosphatase VH1, and the oxidoreductase G4 as bona fide LB components. After reaching the cytosol, F17 was degraded in a proteasome-dependent manner. Proteasome activity, and presumably LB disassembly, was required for the immediate immunomodulatory activity of VH1: dephosphorylation of STAT1 to prevent interferon-gamma-mediated antiviral responses. These results reveal a mechanism used by poxviruses to deliver viral enzymes to the host cell cytosol and are likely to facilitate the identification of additional LB-resident viral effectors.

Structure and Substrate-Induced Conformational Changes of the Secondary Citrate/Sodium Symporter CitS Revealed by Electron Crystallography

F. Kebbel; M. Kurz; M. Arheit; M. G. Gruetter; H. Stahlberg 

The secondary Na+/citrate symporter CitS of Klebsiella pneumoniae is the best-characterized member of the 2-hydroxycarboxylate transporter family. The recent projection structure gave insight into its overall structural organization. Here, we present the three-dimensional map of dimeric CitS obtained with electron crystallography. Each monomer has 13 alpha-helical transmembrane segments; six are organized in a distal helix cluster and seven in the central dimer interface domain. Based on structural analyses and comparison to VcINDY, we propose a molecular model for CitS, assign the helices, and demonstrate the internal structural symmetry. We also present projections of CitS in several conformational states induced by the presence and absence of sodium and citrate as substrates. Citrate binding induces a defined movement of alpha helices within the distal helical cluster. Based on this, we propose a substrate translocation site and conformational changes that are in agreement with the transport model of “alternating access”.

Growth of Large and Highly Ordered 20 Crystals of a K+ Channel, Structural Role of Lipidic Environment

R. De Zorzi; W. V. Nicholson; J-M. Guigner; F. Erne-Brand; H. Stahlberg et al. 

2D crystallography has proven to be an excellent technique to determine the 3D structure of membrane proteins. Compared to 3D crystallography, it has the advantage of visualizing the protein in an environment closer to the native one. However, producing good 2D crystals is still a challenge and little statistical knowledge can be gained from literature. Here, we present a thorough screening of 2D crystallization conditions for a prokaryotic inwardly rectifying potassium channel (>130 different conditions). Key parameters leading to very large and well-organized 2D crystals are discussed. In addition, the problem of formation of multilayers during the growth of 2D crystals is also addressed. An intermediate resolution projection map of KirBac3.1 at 6 A is presented, which sheds (to our knowledge) new light on the structure of this channel in a lipid environment.

Bridging from conventional marketed immediate release formulations to new tamper resistant alternatives

H. Stahlberg; M. Brett; J. Ossig; S. Schwier; A. Philipp 

Image Processing of 2D Crystal Images

Cryo-electron tomography reveals four-membrane architecture of the Plasmodium apicoplast

L. Lemgruber; M. Kudryashev; C. Dekiwadia; D. T. Riglar; J. Baum et al. 

Background: The apicoplast is a plastid organelle derived from a secondary endosymbiosis, containing biosynthetic pathways essential for the survival of apicomplexan parasites. The Toxoplasma apicoplast clearly possesses four membranes but in related Plasmodium spp. the apicoplast has variably been reported to have either three or four membranes.Methods: Cryo-electron tomography was employed to image merozoites of Plasmodium falciparum and Plasmodium berghei frozen in their near-native state. Three-dimensional reconstructions revealed the number of apicoplast membranes and the association of the apicoplast with other organelles. Routine transmission electron microscopy of parasites preserved by high-pressure freezing followed by freeze substitution techniques was also used to analyse apicoplast morphology.Results: Cryo-preserved parasites showed clearly four membranes surrounding the apicoplast. A wider gap between the second and third apicoplast membranes was frequently observed. The apicoplast was found in close proximity to the nucleus and to the rhoptries. The apicoplast matrix showed ribosome-sized particles and membranous whorls.Conclusions: The Plasmodium apicoplast possesses four membranes, as do the apicoplasts of other apicomplexan parasites. This is consistent with a four-membraned secondary endosymbiotic plastid ancestor.

In situ structural analysis of the Yersinia enterocolitica injectisome

M. Kudryashev; M. Stenta; S. Schmelz; M. Amstutz; U. Wiesand et al. 

Injectisomes are multi-protein transmembrane machines allowing pathogenic bacteria to inject effector proteins into eukaryotic host cells, a process called type III secretion. Here we present the first three-dimensional structure of Yersinia enterocolitica and Shigella flexneri injectisomes in situ and the first structural analysis of the Yersinia injectisome. Unexpectedly, basal bodies of injectisomes inside the bacterial cells showed length variations of 20%. The in situ structures of the Y. enterocolitica and S. flexneri injectisomes had similar dimensions and were significantly longer than the isolated structures of related injectisomes. The crystal structure of the inner membrane injectisome component YscD appeared elongated compared to a homologous protein, and molecular dynamics simulations documented its elongation elasticity. The ring-shaped secretin YscC at the outer membrane was stretched by 30-40% in situ, compared to its isolated liposome-embedded conformation. We suggest that elasticity is critical for some two-membrane spanning protein complexes to cope with variations in the intermembrane distance.

Molecular assembly of the aerolysin pore reveals a swirling membrane-insertion mechanism

M. T. Degiacomi; I. Lacovache; L. Pernot; M. Chami; M. Kudryashev et al. 

Aerolysin is the founding member of a superfamily of (beta-pore-forming toxins whose pore structure is unknown. We have combined X-ray crystallography, cryo-EM, molecular dynamics and computational modeling to determine the structures of aerolysin mutants in their monomeric and heptameric forms, trapped at various stages of the pore formation process. A dynamic modeling approach based on swarm intelligence was applied, whereby the intrinsic flexibility of aerolysin extracted from new X-ray structures was used to fully exploit the cryo-EM spatial restraints. Using this integrated strategy, we obtained a radically new arrangement of the prepore conformation and a near-atomistic structure of the aerolysin pore, which is fully consistent with all of the biochemical data available so far. Upon transition from the prepore to pore, the aerolysin heptamer shows a unique concerted swirling movement, accompanied by a vertical collapse of the complex, ultimately leading to the insertion of a transmembrane beta-barrel.

Thermal Unfolding of a Mammalian Pentameric Ligand-gated Ion Channel Proceeds at Consecutive, Distinct Steps

M. B. Tol; C. Deluz; G. Hassaine; A. Graff; H. Stahlberg et al. 

Pentameric ligand-gated ion channels (LGICs) play an important role in fast synaptic signal transduction. Binding of agonists to the beta-sheet-structured extracellular domain opens an ion channel in the transmembrane alpha-helical region of the LGIC. How the structurally distinct and distant domains are functionally coupled for such central transmembrane signaling processes remains an open question. To obtain detailed information about the stability of and the coupling between these different functional domains, we analyzed the thermal unfolding of a homopentameric LGIC, the 5-hydroxytryptamine receptor (ligand binding, secondary structure, accessibility of Trp and Cys residues, and aggregation), in plasma membranes as well as during detergent extraction, purification, and reconstitution into artificial lipid bilayers. We found a large loss in thermostability correlating with the loss of the lipid bilayer during membrane solubilization and purification. Thermal unfolding of the 5-hydroxytryptamine receptor occurred in consecutive steps at distinct protein locations. A loss of ligand binding was detected first, followed by formation of different transient low oligomeric states of receptor pentamers, followed by partial unfolding of helical parts of the protein, which finally lead to the formation receptor aggregates. Structural destabilization of the receptor in detergents could be partially reversed by reconstituting the receptor into lipid bilayers. Our results are important because they quantify the stability of LGICs during detergent extraction and purification and can be used to create stabilized receptor proteins for structural and functional studies.

Automation of image processing in electron crystallography

Merging of image data in electron crystallography

RNAi Screening Reveals Proteasome- and Cullin3-Dependent Stages in Vaccinia Virus Infection

J. Mercer; B. Snijder; R. Sacher; C. Burkard; C. K. E. Bleck et al. 

A two-step, automated, high-throughput RNAi silencing screen was used to identify host cell factors required during vaccinia virus infection. Validation and analysis of clustered hits revealed previously unknown processes during virus entry, including a mechanism for genome uncoating. Viral core proteins were found to be already ubiquitinated during virus assembly. After entering the cytosol of an uninfected cell, the viral DNA was released from the core through the activity of the cell’s proteasomes. Next, a Cullin3-based ubiquitin ligase mediated a further round of ubiquitination and proteasome action. This was needed in order to initiate viral DNA replication. The results accentuate the value of large-scale RNAi screens in providing directions for detailed cell biological investigation of complex pathways. The list of cell functions required during poxvirus infection will, moreover, provide a resource for future virus-host cell interaction studies and for the discovery of antivirals.

The application of graphene as a sample support in transmission electron microscopy

R. S. Pantelic; J. C. Meyer; U. Kaiser; H. Stahlberg 

Transmission electron microscopy has witnessed rampant development and surging point resolution over the past few years. The improved imaging performance of modern electron microscopes shifts the bottleneck for image contrast and resolution to sample preparation. Hence, it is increasingly being realized that the full potential of electron microscopy will only be realized with the optimization of current sample preparation techniques. Perhaps the most recognized issues are background signal and noise contributed by sample supports, sample charging and instability. Graphene provides supports of single atom thickness, extreme physical stability, periodic structure, and ballistic electrical conductivity. As an increasing number of applications adapting graphene to their benefit emerge, we discuss the unique capabilities afforded by the use of graphene as a sample support for electron microscopy. (c) 2012 Elsevier Ltd. All rights reserved.

Structural basis for chirality and directional motility of Plasmodium sporozoites

M. Kudryashev; S. Muenter; L. Lemgruber; G. Montagna; H. Stahlberg et al. 

Plasmodium sporozoites can move at high speed for several tens of minutes, which is essential for the initial stage of a malaria infection. The crescent-shaped sporozoites move on 2D substrates preferably in the same direction on circular paths giving raise to helical paths in 3D matrices. Here we determined the structural basis that underlies this type of movement. Immature, non-motile sporozoites were found to lack the subpellicular network required for obtaining the crescent parasite shape. In vitro, parasites moving in the favoured direction move faster and more persistent than the few parasites that move in the opposite direction. Photobleaching experiments showed that sporozoites flip their ventral side up when switching the direction of migration. Cryo-electron tomography revealed a polarized arrangement of microtubules and polar rings towards the substrate in Plasmodium sporozoites, but not in the related parasite Toxoplasma gondii. As aconsequence, secretory vesicles, which release proteins involved in adhesion, migration and invasion at the front end of the parasite, are delivered towards the substrate. The resulting chiral structure of the parasite appears to determine the unique directionality of movement and could explain how the sporozoite achieves rapid and sustained directional motility in the absence of external stimuli.

Characterization of the motion of membrane proteins using high-speed atomic force microscopy

I. Casuso; J. Khao; M. Chami; P. Paul-Gilloteaux; M. Husain et al. 

For cells to function properly(1), membrane proteins must be able to diffuse within biological membranes. The functions of these membrane proteins depend on their position and also on protein-protein and protein-lipid interactions(2). However, so far, it has not been possible to study simultaneously the structure and dynamics of biological membranes. Here, we show that the motion of unlabelled membrane proteins can be characterized using high-speed atomic force microscopy(3). We find that the molecules of outer membrane protein F (OmpF) are widely distributed in the membrane as a result of diffusion-limited aggregation, and while the overall protein motion scales roughly with the local density of proteins in the membrane, individual protein molecules can also diffuse freely or become trapped by protein-protein interactions. Using these measurements, and the results of molecular dynamics simulations, we determine an interaction potential map and an interaction pathway for a membrane protein, which should provide new insights into the connection between the structures of individual proteins and the structures and dynamics of supramolecular membranes.

Limiting factors in single particle cryo electron tomography

M. Kudryashev; D. Castano-Diez; H. Stahlberg 

Modern methods of cryo electron microscopy and tomography allow visualization of protein nanomachines in their native state at the nanometer scale. Image processing methods including sub-volume averaging applied to repeating macromolecular elements within tomograms allow exploring their structures within the native context of the cell, avoiding the need for protein isolation and purification. Today, many different data acquisition protocols and software solutions are available to researchers to determine average structures of macromolecular complexes and potentially to classify structural intermediates. Here, we list the density maps reported in the literature, and analyze each structure for the chosen instrumental settings, sample conditions, main processing steps, and obtained resolution. We present conclusions that identify factors currently limiting the resolution gained by this approach.

Mus81-Mms4 Functions as a Single Heterodimer To Cleave Nicked Intermediates in Recombinational DNA Repair

E. K. Schwartz; W. D. Wright; K. T. Ehmsen; J. E. Evans; H. Stahlberg et al. 

The formation of crossovers is a fundamental genetic process. The XPF-family endonuclease Mus81-Mms4 (Eme1) contributes significantly to crossing over in eukaryotes. A key question is whether Mus81-Mms4 can process Holliday junctions that contain four uninterrupted strands. Holliday junction cleavage requires the coordination of two active sites, necessitating the assembly of two Mus81-Mms4 heterodimers. Contrary to this expectation, we show that Saccharomyces cerevisiae Mus81-Mms4 exists as a single heterodimer both in solution and when bound to DNA substrates in vitro. Consistently, immunoprecipitation experiments demonstrate that Mus81-Mms4 does not multimerize in vivo. Moreover, chromatin-bound Mus81-Mms4 does not detectably form higher-order multimers. We show that Cdc5 kinase activates Mus81-Mms4 nuclease activity on 3′ flaps and Holliday junctions in vitro but that activation does not induce a preference for Holliday junctions and does not induce multimerization of the Mus81-Mms4 heterodimer. These data support a model in which Mus81-Mms4 cleaves nicked recombination intermediates such as displacement loops (D-loops), nicked Holliday junctions, or 3′ flaps but not intact Holliday junctions with four uninterrupted strands. We infer that Mus81-dependent crossing over occurs in a noncanonical manner that does not involve the coordinated cleavage of classic Holliday junctions.

Dynamo: A flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments

D. Castano-Diez; M. Kudryashev; M. Arheit; H. Stahlberg 

Dynamo is a new software package for subtomogram averaging of cryo Electron Tomography (cryo-ET) data with three main goals: first, Dynamo allows user-transparent adaptation to a variety of high-performance computing platforms such as CPUs or CPU clusters. Second, Dynamo implements user-friendliness through GUI interfaces and scripting resources. Third, Dynamo offers user-flexibility through a plugin API. Besides the alignment and averaging procedures, Dynamo includes native tools for visualization and analysis of results and data, as well as support for third party visualization software, such as Chimera UCSF or EMAN2. As a demonstration of these functionalities, we studied bacterial flagellar motors and showed automatically detected classes with absent and present C-rings.Subtomogram averaging is a common task in current cryo-ET pipelines, which requires extensive computational resources and follows a well-established workflow. However, due to the data diversity, many existing packages offer slight variations of the same algorithm to improve results. One of the main purposes behind Dynamo is to provide explicit tools to allow the user the insertion of custom designed procedures – or plugins – to replace or complement the native algorithms in the different steps of the processing pipeline for subtomogram averaging without the burden of handling parallelization. Custom scripts that implement new approaches devised by the user are integrated into the Dynamo data management system, so that they can be controlled by the GUI or the scripting capacities.Dynamo executables do not require licenses for third party commercial software. Sources, executables and documentation are freely distributed on http://www.dynamo-em.org. (C) 2012 Elsevier Inc. All rights reserved.

Assessing the benefits of focal pair cryo-electron tomography

M. Kudryashev; H. Stahlberg; D. Castano-Diez 

Cryo electron tomography provides nanometer-scale information on biological matter preserved in a close-to native state. The resolution of tomograms and structures resolved by sub-tomogram averaging is typically limited by the contrast transfer function of the electron microscope, which is especially critical for thick samples. Here, we report a method to increase the attainable resolution by recording tomographic ‘focal pairs’, which are pairs of tilt series of the same object acquired in complementary defocus conditions. Low defocus imaging provides high resolution at low contrast, while high defocus imaging yields high contrast at the price of limited resolution. Quantitative assessment of the quality of lipid bilayer reconstructions in the resulting tomograms demonstrates stable resolution preservation beyond 3 nm for cells thicker than 500 nm. Further, in computational simulations on synthetic datasets we show the applicability of the method to sub-tomogram averaging, demonstrating its potential for achieving higher resolution. (C) 2011 Elsevier Inc. All rights reserved.

Structure and Function of Purified Monoclonal Antibody Dimers Induced by Different Stress Conditions

R. Paul; A. Graff-Meyer; H. Stahlberg; M. E. Lauer; A. C. Rufer et al. 

To investigate structure and function of different monoclonal antibody (MAb) dimers.MAb dimers were induced by process-related, low pH and UV light stress. Dimers were isolated and purified by chromatography and extensively characterized by biochemical, structural and functional methods.Highly purified dimer forms were obtained which enabled detailed characterization. Dimers induced by process stress were associated by a single non-covalent interaction site between two Fab domains in a characteristic “bone-like” structure observed in Transmission Electron Microscopy (TEM). These dimers showed reduced potency and antigen binding affinity. Low pH stress generated more stable but also non-covalently associated dimers without chemical alterations in a typical “closed” conformation according to TEM. These dimer species were more compact and more hydrophobic as dimers induced by process stress. They showed bioactivity and antigen binding affinity similar to the native monomer. Light-induced dimers, exhibiting various different conformations, were the most stable dimers with various chemical modifications leading to a broad range in size, charge and hydrophobicity. These dimers fully lost bioactivity and antigen binding affinity.The use of highly purified MAb dimers and a panel of characterizations methods enabled to obtain a clear picture about molecular architecture and function of dimers.

Projection Structure of the Secondary Citrate/Sodium Symporter CitS at 6 angstrom Resolution by Electron Crystallography

F. Kebbel; M. Kurz; M. G. Gruetter; H. Stahlberg 

CitS from Klebsiella pneumoniae acts as a secondary symporter of citrate and sodium ions across the inner membrane of the host. The protein is the best characterized member of the 2-hydroxycarboxylate transporter family, while no experimental structural information at sub-nanometer resolution is available on this class of membrane proteins. Here, we applied electron crystallography to two-dimensional crystals of CitS. Carbon-film-adsorbed tubular two-dimensional crystals were studied by cryo-electron microscopy, producing the 6-angstrom-resolution projection structure of the membrane-embedded protein. In the p22(1)2(1)-symmetrized projection map, the predicted dimeric structure is clearly visible. Each monomeric unit can tentatively be interpreted as being composed of 11 transmembrane alpha-helices. In projection, CitS shows a high degree of structural similarity to NhaP1, the Na+/H+ antiporter of Methanococcus jannaschii. We discuss possible locations for the dimer interface and models for the helical arrangements and domain organizations of the symporter based on existing models. (C) 2012 Elsevier Ltd. All rights reserved.

Iterative Transform Algorithms for 3D Reconstruction of 2D Crystals

X. Z. –; Y. C. –; O. H. –; H. Stahlberg 

Connecting mu-fluidics to electron microscopy

S. Kemmerling; J. Ziegler; G. Schweighauser; S. A. Arnold; D. Giss et al. 

A versatile methodology for electron microscopy (EM) grid preparation enabling total content sample analysis is presented. A microfluidic-dialysis conditioning module to desalt or mix samples with negative stain solution is used, combined with a robotic writing table to micro-pattern the EM grids. The method allows heterogeneous samples of minute volumes to be processed at physiological pH for structure and mass analysis, and allows the preparation characteristics to be finely tuned. (C) 2011 Elsevier Inc. All rights reserved.

Ionic liquids as matrices in microfluidic sample deposition for high-mass matrix-assisted laser desorption/ionization mass spectrometry

S. Weidmann; S. Kemmerling; S. Maedler; H. Stahlberg; T. Braun et al. 

Sample preparation for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) via a microfluidic deposition device using ionic liquid matrices addresses several problems of standard protocols with crystalline matrices, such as the heterogeneity of sample spots due to the co-crystallization of sample and matrix and the limited capability for high-throughput analysis. Since ionic Liquid matrices do not solidify during the measurement, the resulting sample spots are homogeneous. The use of these matrices is also beneficial for automated sample preparation, since crystallization of the matrix is avoided and, thus, no clogging of the spotting device can occur. The applicability of ionic liquids to the analysis of biomolecules with high molecular weights, up to approximate to 1 MDa is shown, as well as a good sensitivity [5fmol] for recombinant human fibronectin, a protein with a molecular weight of 226 kDa. Microfluidic sample deposition of proteins with high molecular weights will, in the future, allow parallel sample preparation for MALDI-MS and for electron microscopy.

Complete Lateral and Angular Diffusion and Protein-Protein Interaction Description of a Membrane Protein

I. Casuso; J-P. Duneau; M. Chami; P. Paul-Gilloteaux; M. Husain et al. 

Interactions of CCR5, the Main HIV Coreceptor, with Rantes and Other Ligands

S. Morin; L. Nisius; M. Wiktor; F. Kebbel; H. Stahlberg et al. 

Rad51 paralogues Rad55-Rad57 balance the antirecombinase Srs2 in Rad51 filament formation

J. Liu; L. Renault; X. Veaute; F. Fabre; H. Stahlberg et al. 

Homologous recombination is a high-fidelity DNA repair pathway. Besides a critical role in accurate chromosome segregation during meiosis, recombination functions in DNA repair and in the recovery of stalled or broken replication forks to ensure genomic stability. In contrast, inappropriate recombination contributes to genomic instability, leading to loss of heterozygosity, chromosome rearrangements and cell death. The RecA/UvsX/RadA/Rad51 family of proteins catalyses the signature reactions of recombination, homology search and DNA strand invasion(1,2). Eukaryotes also possess Rad51 paralogues, whose exact role in recombination remains to be defined(3). Here we show that the Saccharomyces cerevisiae Rad51 paralogues, the Rad55-Rad57 heterodimer, counteract the antirecombination activity of the Srs2 helicase. The Rad55-Rad57 heterodimer associates with the Rad51-single-stranded DNA filament, rendering it more stable than a nucleoprotein filament containing Rad51 alone. The Rad51-Rad55-Rad57 co-filament resists disruption by the Srs2 antirecombinase by blocking Srs2 translocation, involving a direct protein interaction between Rad55-Rad57 and Srs2. Our results demonstrate an unexpected role of the Rad51 paralogues in stabilizing the Rad51 filament against a biologically important antagonist, the Srs2 antirecombination helicase. The biological significance of this mechanism is indicated by a complete suppression of the ionizing radiation sensitivity of rad55 or rad57 mutants by concomitant deletion of SRS2, as expected for biological antagonists. We propose that the Rad51 presynaptic filament is a meta-stable reversible intermediate, whose assembly and disassembly is governed by the balance between Rad55-Rad57 and Srs2, providing a key regulatory mechanism controlling the initiation of homologous recombination. These data provide a paradigm for the potential function of the human RAD51 paralogues, which are known to be involved in cancer predisposition and human disease.

Oxidative Doping Renders Graphene Hydrophilic, Facilitating Its Use As a Support in Biological TEM

R. S. Pantelic; J. W. Suk; Y. Hao; R. S. Ruoff; H. Stahlberg 

Graphene represents the first practical realization of crystalline supports in biological transmission electron microscopy (TEM) since their introduction over 30 years ago. The high transparency, minimal inelastic cross-section, and electrical conductivity of graphene are highly desirable characteristics for a TEM support. However, without a suitable method for rendering graphene supports, hydrophilic applications are limited. This work describes the in situ functionalization of graphene with minimal structural degradation, rendering TEM supports sufficiently hydrophilic for the mounting of biological samples.

Interaction of complexes I, III, and IV within the bovine respirasome by single particle cryoelectron tomography

N. V. Dudkina; M. Kudryashev; H. Stahlberg; E. J. Boekema 

The respirasome is a multisubunit supercomplex of the respiratory chain in mitochondria. Here we report the 3D reconstruction of the bovine heart respirasome, composed of dimeric complex III and single copies of complex I and IV, at about 2.2-nm resolution, determined by cryoelectron tomography and subvolume averaging. Fitting of X-ray structures of single complexes I, III2, and IV with high fidelity allows interpretation of the model at the level of secondary structures and shows how the individual complexes interact within the respirasome. Surprisingly, the distance between cytochrome c binding sites of complexes III2 and IV is about 10 nm. Modeling indicates a loose interaction between the three complexes and provides evidence that lipids are gluing them at the interfaces.

Automatic recovery of missing amplitudes and phases in tilt-limited electron crystallography of two-dimensional crystals

B. R. Gipson; D. J. Masiel; N. D. Browning; J. Spence; K. Mitsuoka et al. 

Electron crystallography of 2D protein crystals provides a powerful tool for the determination of membrane protein structure. In this method, data is acquired in the Fourier domain as randomly sampled, uncoupled, amplitudes and phases. Due to physical constraints on specimen tilting, those Fourier data show a vast un-sampled “missing cone” of information, producing resolution loss in the direction perpendicular to the membrane plane. Based on the flexible language of projection onto sets, we provide a full solution for these problems with a projective constraint optimization algorithm that, for sufficiently oversampled data, produces complete recovery of unmeasured data in the missing cone. We apply this method to an experimental data set of Bacteriorhodopsin and show that, in addition to producing superior results compared to traditional reconstruction methods, full, reproducible, recovery of the missing cone from noisy data is possible. Finally, we present an automatic implementation of the refinement routine as open source, freely distributed, software that will be included in our 2dx software package.

Polymer-based cell-free expression of ligand-binding family B G-protein coupled receptors without detergents

C. Klammt; M. H. Perrin; I. Maslennikov; L. Renault; M. Krupa et al. 

G-protein coupled receptors (GPCRs) constitute the largest family of intercellular signaling molecules and are estimated to be the target of more than 50% of all modern drugs. As with most integral membrane proteins (IMPs), a major bottleneck in the structural and biochemical analysis of GPCRs is their expression by conventional expression systems. Cell-free (CF) expression provides a relatively new and powerful tool for obtaining preparative amounts of IMPs. However, in the case of GPCRs, insufficient homogeneity of the targeted protein is a problem as the in vitro expression is mainly done with detergents, in which aggregation and solubilization difficulties, as well as problems with proper folding of hydrophilic domains, are common. Here, we report that using CF expression with the help of a fructose-based polymer, NV10 polymer (NVoy), we obtained preparative amounts of homogeneous GPCRs from the three GPCR families. We demonstrate that two GPCR B family members, corticotrophin-releasing factor receptors 1 and 2 beta are not only solubilized in NVoy but also have functional ligand-binding characteristics with different agonists and antagonists in a detergent-free environment as well. Our findings open new possibilities for functional and structural studies of GPCRs and IMPs in general.

Graphene: Substrate preparation and introduction

R. S. Pantelic; J. W. Suk; C. W. Magnuson; J. C. Meyer; P. Wachsmuth et al. 

This technical note describes the transfer of continuous, single-layer, pristine graphene to standard Quantifoil TEM grids. We compare the transmission properties of pristine graphene substrates to those of graphene oxide and thin amorphous carbon substrates. Positively stained DNA imaged across amorphous carbon is typically indiscernible and requires metal shadowing for sufficient contrast. However, in a practical illustration of the new substrates properties, positively stained DNA is imaged across pristine graphene in striking contrast without the need of metal shadowing. We go onto discuss technical considerations and the potential applications of pristine graphene substrates as well as their ongoing development. (C) 2010 Elsevier Inc. All rights reserved.

3D Reconstruction of 2D Crystals

X. Zeng; J. E. Glover; O. Hughes; H. Stahlberg 

High Resolution three-dimensional (3D) reconstruction of several proteins has been achieved from two-dimensional (2D) crystals by electron crystallography structure determination. However, for badly ordered 2D crystals, especially non-flat crystals, Fourierfiltering based methods fail, while single particle processing approaches can produce reconstructions of superior resolution by aligning particles in 3D space. We have investigated a single particle processing approach combined with the crystallographic method to generate images centered on the unit cells of 2D crystal images. The implemented software uses the predictive lattice node tracking in 2dx/ MRC software to extract particles from the microscope images. These particles are then subjected to a local contrast transfer function (CTF) correction. The tilt geometry obtained in the 2dx software is used to initialize the Euler angles, which along with translations are then refined by a single particle processing approach. Finally, iterative transform algorithms, namely the error reduction algorithm and the hybrid input-output algorithm, are applied to retrieve missing information in the previously obtained 3D reconstruction. Compared with conventional single particle processing for randomly oriented particles, the required computational costs are greatly reduced as the 2D crystals restrict the parameter search space. Preliminary results from a 3D reconstruction of the membrane protein GlpF suggest that the iterative transform process improves 3D resolution.

Filling the missing cone: Automatic recovery of data in tilt-limited microscopy

H. Stahlberg; X. Zeng; D. J. Masiel; N. Browning; J. Spence et al. 

An optical and microPET assessment of thermally-sensitive liposome biodistribution in the Met-1 tumor model: Importance of formulation

E. E. Paoli; D. E. Kruse; J. W. Seo; H. Zhang; A. Kheirolomoom et al. 

The design of delivery vehicles that are stable in circulation but can be activated by exogenous energy sources is challenging. Our goals are to validate new imaging methods for the assessment of particle stability, to engineer stable and activatable particles and to assess accumulation of a hydrophilic model drug in an orthotopic tumor. Here, liposomes were injected into the tail vein of FVB mice containing bilateral Met-1 tumors and imaged in vivo using microPET and optical imaging techniques. Cryo-electron microscopy was applied to assess particle shape prior to injection, ex vivo fluorescence images of dissected tissues were acquired, excised tissue was further processed with a cell-digest preparation and assayed for fluorescence. We find that for a stable particle, in vivo tumor images of a hydrophilic model drug were highly correlated with PET images of the particle shell and ex vivo fluorescence images of processed tissue, R-2 = 0.95 and R-2 = 0.99 respectively. We demonstrate that the accumulation of a hydrophilic model drug is increased by up to 177 fold by liposomal encapsulation, as compared to accumulation of the drug at 24 hours. (C) 2009 Elsevier B.V. All rights reserved.

Preparation of 2D Crystals of Membrane Proteins for High-Resolution Electron Crystallography Data Collection

3D Reconstruction from 2D Crystal Image and Diffraction Data

High-resolution low-dose scanning transmission electron microscopy

J. P. Buban; Q. Ramasse; B. Gipson; N. D. Browning; H. Stahlberg 

During the past two decades instrumentation in scanning transmission electron microscopy (STEM) has pushed toward higher intensity electron probes to increase the signal-to-noise ratio of recorded images. While this is suitable for robust specimens, biological specimens require a much reduced electron dose for high-resolution imaging. We describe here protocols for low-dose STEM image recording with a conventional field-emission gun STEM, while maintaining the high-resolution capability of the instrument. Our findings show that a combination of reduced pixel dwell time and reduced gun current can achieve radiation doses comparable to low-dose TEM.

Coassembly of Mgm1 isoforms requires cardiolipin and mediates mitochondrial inner membrane fusion

R. M. DeVay; L. Dominguez-Ramirez; L. L. Lackner; S. Hoppins; H. Stahlberg et al. 

Two dynamin-related protein (DRP) families are essential for fusion of the outer and inner mitochondrial membranes, Fzo1 (yeast)/Mfn1/Mfn2 (mammals) and Mgm1 (yeast)/Opa1 (mammals), respectively. Fzo1/Mfns possess two medial transmembrane domains, which place their critical GTPase and coiled-coil domains in the cytosol. In contrast, Mgm1/Opa1 are present in cells as long (l) isoforms that are anchored via the N terminus to the inner membrane, and short (s) isoforms were predicted to be soluble in the intermembrane space. We addressed the roles of Mgm1 isoforms and how DRPs function in membrane fusion. Our analysis indicates that in the absence of a membrane, l- and s-Mgm1 both exist as inactive GTPase monomers, but that together in trans they form a functional dimer in a cardiolipin-dependent manner that is the building block for higher-order assemblies.

Structural variability of edge dislocations in a SrTiO(3) low-angle [001] tilt grain boundary

J. P. Buban; M. Chi; D. J. Masiel; J. P. Bradley; B. Jiang et al. 

Using a spherical aberration (Cs)-corrected scanning transmission electron microscopy (STEM) and electron energy-loss spectroscopy (EELS), we Investigated a 6 degrees low-angle [001] tilt grain boundary in SrTiO(3). The enhanced spatial resolution of the aberration corrector leads to the observation of a number Of Structural variations ill the edge dislocations along the grain boundary that neither resemble the standard edge dislocations nor partial dislocations for SrTiO(3). Although there appear to be many variants in the structure that can be interpreted as compositional effects, three main classes of core structure are found to be prominent. From EELS analysis, these classifications seem to be related to Sr deficiencies, with the final variety of the cores being consistent with an embedded TiO(x) rocksalt-like structure.

Crystal Structures of Limulus SAP-Like Pentraxin Reveal Two Molecular Aggregations

A. K. Shrive; I. Burns; H-T. Chou; H. Stahlberg; P. B. Armstrong et al. 

The serum-amyloid-P-component-like pentraxin from Limulus polyphemus, a recently discovered pentraxin species and important effector protein of the hemolymph immune system, displays two distinct doubly stacked cyclic molecular aggregations, heptameric and octameric. The refined three-dimensional structures determined by X-ray crystallography, both based on the same cDNA sequence, show that each aggregate is constructed from a similar dimer of protomers, which is repeated to make up the ring structure. The native The native octameric form has been refined at a resolution of 3 angstrom, the native heptameric form at 2.3 angstrom, and the phosphoethanolamine (PE)-bound octameric form at 2.7 angstrom. The existence of the hitherto undescribed heptameric form was confirmed by single-particle analysis using cryo-electron microscopy. In the native structures, the calcium-binding site is similar to that in human pentraxins, with two calcium ions bound in each subunit. Upon binding PE, however, each subunit binds a third calcium ion, with all three calcium ions contributing to the binding and orientation of the bound phosphate group within the ligand-binding pocket. While the phosphate is well-defined in the electron density, the ethanolamine group is poorly defined, suggesting structural and binding variabilities of this group. Although sequence homology with human serum amyloid P component is relatively low, structural homology is high, with very similar overall folds and a common affinity for PE. This is due, in part, to a “topological” equivalence of side-chain position. Identical side chains that are important in both function and fold, from different regions of the sequence in human and Limulus structures, occupy similar space within the overall subunit fold. Sequence and structure alignment, based on the refined three-dimensional structures presented here and the known horseshoe crab pentraxin sequences, suggest that adaptation and refinement of C-reactive-protein-mediated immune responses in these ancient creatures lacking antibody-based immunity are based on adaptation by gene duplication. (c) 2009 Elsevier Ltd. All rights reserved.

Membrane activity of a C-reactive protein

J. M. Harrington; H-T. Chou; T. Gutsmann; C. Gelhaus; H. Stahlberg et al. 

C-reactive protein (CRP) from the American horseshoe crab, Limulus polyphemus, exhibits complex membrane activities. Here, we describe the behavior of protein and lipid as CRP interacts with model liposomes and bacterial membranes. Limulus C-reactive protein (L-CRP) forms extended fibrilar structures that encapsulate liposomes in the presence of Ca2+. We have observed structures consistent in size and shape with these fibers bound to the surface of Gram-negative bacteria. The membranes of Limulus CRP-treated bacteria exhibit significantly different mechano-elastic properties than those of untreated bacteria. In vitro, bilayer lipids undergo a rigidification and reorganization of small domains. We suggest that these interactions reflect the protein’s role as a primary defense molecule, functioning in the entrapment and killing of potential pathogens. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

2007 annual progress report synopsis of the Center for Structures of Membrane Proteins

R. M. Stroud; S. Choe; J. Holton; H. R. Kaback; W. Kwiatkowski et al. 

A synopsis of the 2007 annual progress report for the Center for Structures of Membrane Proteins, a specialized center of the Protein Structure Initiative.

Low-dose aberration corrected cryo-electron microscopy of organic specimens

J. E. Evans; C. Hetherington; A. Kirkland; L-Y. Chang; H. Stahlberg et al. 

Spherical aberration (C-s) correction in the transmission electron microscope has enabled sub-angstrom resolution imaging of inorganic materials. To achieve similar resolution for radiation-sensitive organic materials requires the microscope to be operated under hybrid conditions: low electron dose illumination of the specimen at liquid nitrogen temperature and low defocus values. Initial images from standard inorganic and organic test specimens have indicated that under these conditions C-s-correction can provide a significant improvement in resolution (to less than 0.16 nm) for direct imaging of organic samples. (C) 2008 Published by Elsevier B.V.

Bridging across length scales: Multi-scale ordering of supported lipid bilayers via lipoprotein self-assembly and surface patterning

M. S. Vinchurkar; D. A. Bricarello; J. O. Lagerstedt; J. P. Buban; H. Stahlberg et al. 

We show that a two-step process, involving spontaneous self-assembly of lipids and apolipoproteins and surface patterning, produces single, supported lipid bilayers over two discrete and independently adjustable length scales. Specifically, an aqueous phase incubation of DMPC vesicles with purified apolipoprotein A-I results in the reconstitution of high density lipoprotein (rHDL), wherein nanoscale clusters of single lipid bilayers are corralled by the protein. Adsorption of these discoidal particles to clean hydrophilic glass (or silicon) followed by direct exposure to a spatial pattern of short-wavelength UV radiation directly produces microscopic patterns of nanostructured bilayers. Alternatively, simple incubation of aqueous phase rHDL with a chemically patterned hydrophilic/hydrophobic surface produces a novel compositional pattern, caused by an increased affinity for adsorption onto hydrophilic regions relative to the surrounding hydrophobic regions. Further, by simple chemical denaturation of the boundary protein, nanoscale compartmentalization can be selectively erased, thus producing patterns of laterally fluid, lipid bilayers structured solely at the mesoscopic length scale. Since these aqueous phase microarrays of nanostructured lipid bilayers allow for membrane proteins to be embedded within single nanoscale bilayer compartments, they present a viable means of generating high-density membrane protein arrays. Such a system would permit in-depth elucidation of membrane protein structure-function relationships and the consequences of membrane compartmentalization on lipid dynamics.

2dx – Automated 3D structure reconstruction from 2D crystal data

B. Gipson; X. Zeng; H. Stahlberg 

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

Membrane pore formation by pentraxin proteins from Limulus, the American horseshoe crab

J. M. Harrington; H-T. Chou; T. Gutsmann; C. Gelhaus; H. Stahlberg et al. 

The pentraxins are a family of highly conserved plasma proteins of metazoans known to function in immune defence. The canonical members, C-reactive protein and serum amyloid P component, have been identified in arthropods and humans. Mammalian pentraxins are known to bind lipid bilayers, and a pentraxin representative from the American horseshoe crab, Limulus polyphemus, binds and permeabilizes mammalian erythrocytes. Both activities are Ca2+-dependent. Utilizing model liposomes and planar lipid bilayers, in the present study we have investigated the m ernbrane-active properties of the three pentraxin representatives from Limulus and show that all of the Limulus pentraxins permeabilize lipid bilayers. Mechanistically, Linudils C-reactive protein forms transmembrane pores in asymmetric planar lipid bilayers that mimic the outer membrane of Gram-negative bacteria and exhibits a Ca2+-independent form of membrane binding that may be sufficient for pore formation.

The fold of alpha-synuclein fibrils

M. Vilar; H-T. Chou; T. Luehrs; S. K. Maji; D. Riek-Loher et al. 

The aggregation of proteins into amyloid fibrils is associated with several neurodegenerative diseases. In Parkinson’s disease it is believed that the aggregation of a-synuclein (alpha-syn) from monomers by intermediates into amyloid fibrils is the toxic disease-causative mechanism. Here, we studied the structure of a-syn in its amyloid state by using various biophysical approaches. Quenched hydrogen/deuterium exchange NMR spectroscopy identified five beta-strands within the fibril core comprising residues 35-96 and solid-state NMR data from amyloid fibrils comprising the fibril core residues 30-110 confirmed the presence of beta-sheet secondary structure. The data suggest that beta 1-strand interacts with beta 2, beta 2 with beta 3, beta 3 with beta 4, and beta 4 with beta 5. High-resolution cryoelectron microscopy revealed the protofilament boundaries of approximate to 2 x 3.5 nm. Based on the combination of these data and published structural studies, a fold of alpha-syn in the fibrils is proposed and discussed.

Molecular electron microscopy: State of the art and current challenges

H. Stahlberg; T. Walz 

The objective of molecular electron microscopy (EM) is to use etectron microscopes to visualize the structure of biological molecules. This Review provides a brief overview of the methods used in molecular EM, their respective strengths and successes, and current developments that promise an even more exciting future for molecular EM in the structural investigation of proteins and macromolecular complexes, studied in isolation or in the context of cells and tissues.

Automatic lattice determination for two-dimensional crystal images

X. Zeng; B. Gipson; Z. Y. Zheng; L. Renault; H. Stahlberg 

Electron crystallography determines the structure of membrane proteins and other periodic samples by recording either images or diffraction patterns. Computer processing of recorded images requires the determination of the reciprocal lattice parameters in the Fourier transform of the image. We have developed a set of three programs 2dx_peaksearch, 2dx_findlat and 2dx_getlat, which can determine the reciprocal lattice from a Fourier transformation of a 2D crystal image automatically. 2dx_peaksearch determines a list of Fourier peak coordinates from a processed calculated diffraction pattern. These coordinates are evaluated by 2dx_findlat to determine one or more lattices, using a-priori knowledge of the real-space crystal unit cell dimensions, and the sample tilt geometry. If these are unknown, then the program 2dx_getlat can be used to obtain a guess for the unit cell dimensions. These programs are available as part of the 2dx software package for the image processing of 2D crystal images at http://2dx.org. (c) 2007 Elsevier Inc. All rights reserved.

2dx_merge: Data management and merging for 2D crystal images

B. Gipson; X. Zeng; H. Stahlberg 

Electron crystallography of membrane proteins determines the structure of membrane-reconstituted and two-dimensionally (2D) crystallized membrane proteins by low-dose imaging with the transmission electron microscope, and computer image processing. We have previously presented the software system 2dx, for user-friendly image processing of 2D crystal images. Its central component 2dx_image is based on the MRC program suite, and allows the optionally fully automatic processing of one 2D crystal image. We present here the program 2dx_merge, which assists the user in the management of a 2D crystal image processing project, and facilitates the merging of the data from multiple images. The merged dataset can be used as a reference to re-process all images, which usually improves the resolution of the final reconstruction. Image processing and merging can be applied iteratively, until convergence is reached. 2dx is available under the GNU General Public License at http://2dx.org. (c) 2007 Elsevier Inc. All rights reserved.

Electron crystallography of membrane proteins

B. Hankamer; R. Glaeser; H. Stahlberg 

A maximum likelihood approach to two-dimensional crystals

X. Zeng; H. Stahlberg; N. Grigorieff 

Maximum likelihood (ML) processing of transmission electron microscopy images of protein particles can produce reconstructions of superior resolution due to a reduced reference bias. We have investigated a ML processing approach to images centered on the unit cells of two-dimensional (2D) crystal images. The implemented software makes use of the predictive lattice node tracking in the MRC software, which is used to window particle stacks. These are then noise-whitened and subjected to ML processing. Resulting NIL maps are translated into amplitudes and phases for further processing within the 2dx software package. Compared with ML processing for randomly oriented single particles, the required computational costs are greatly reduced as the 2D crystals restrict the parameter search space. The software was applied to images of negatively stained or frozen hydrated 2D crystals of different crystal order. We find that the ML algorithm is not free from reference bias, even though its sensitivity to noise correlation is lower than for pure cross-correlation alignment. Compared with crystallographic processing, the newly developed software yields better resolution for 2D crystal images of lower crystal quality, and it performs equally well for well-ordered crystal images. (c) 2007 Elsevier Inc. All rights reserved.

2dx – User Friendly Image Processing (and Merging) for 2D Crystals

B. Gipson; Zeng; H. Stahlberg; R. Wouts 

The structure of the prokaryotic cyclic nucleotide-modulated potassium channel MIoK1 at 16 angstrom resolution

P-L. Chiu; M. D. Pagel; J. Evans; H-T. Chou; X. Zeng et al. 

The gating ring of cyclic nucleotide-modulated channels is proposed to be either a two-fold symmetric dimer of dinners or a four-fold symmetric tetramer based on high-resolution structure data of soluble cyclic nucleotide-binding domains and functional data on intact channels. We addressed this controversy by obtaining structural data on an intact, full-length, cyclic nucleotide-modulated potassium channel, MloK1, from Mesorhizobium loti, which also features a putative voltage-sensor. We present here the 3D single-particle structure by transmission electron microscopy and the projection map of membrane-reconstituted 2D crystals of MloK1 in the presence of cAMP. Our data show a four-fold symmetric arrangement of the CNBDs, separated by discrete gaps. A homology model for full-length MloK1 suggests a vertical orientation for the CNBDs. The 2D crystal packing in the membrane-embedded state is compatible with the S1-S4 domains in the vertical “up” state.

Oocyte CD9 is enriched on the microvillar membrane and required for normal microvillar shape and distribution

K. E. Runge; J. E. Evans; Z-Y. He; S. Gupta; K. L. McDonald et al. 

Microvilli are found on the surface of many cell types, including the mammalian oocyte, where they are thought to act in initial contact of sperm and oocyte plasma membranes. CD9 is currently the only oocyte protein known to be required for sperm-oocyte fusion. We found CD9 is localized to the oocyte microvillar membrane using transmission electron microscopy (TEM). Scanning electron microscopy (SEM) showed that CD9 null oocytes, which are unable to fuse with sperm, have an altered length, thickness and density of their microvilli. One aspect of this change in morphology was quantified using TEM by measuring the radius of curvature at the microvillar tips. A small radius of curvature is thought to promote fusibility and the radius of curvature of microvillar tips on CD9 wild-type oocytes was found to be half that of the CD9 null oocytes. We found that oocyte CD9 co-immunoprecipitates with two Ig superfamily cis partners, EWI-2 and EWI-F, which could have a role in linking CD9 to the oocyte microvillar actin core. We also examined latrunculin B-treated oocytes, which are known to have reduced fusion ability, and found altered microvillar morphology by SEM and TEM. Our data suggest that microvilli may participate in sperm-oocyte fusion. Microvilli could act as a platform to concentrate adhesion/fusion proteins and/or provide a membrane protrusion with a low radius of curvature. They may also have a dynamic interaction with the sperm that serves to capture the sperm cell and bring it into close contact with the oocyte plasma membrane. (c) 2006 Elsevier Inc. All rights reserved.

Structural and kinetic studies of induced fit in xylulose kinase from Escherichia coli

E. Di Luccio; B. Petschacher; J. Voegtli; H-T. Chou; H. Stahlberg et al. 

The primary metabolic route for D-xylose, the second most abundant sugar in nature, is via the pentose phosphate pathway after a two-step or three-step conversion to xylulose-5-phosphate. Xylulose kinase (XK; EC 2.7.1.17) phosphorylates D-xylulose, the last step in this conversion. The apo and D-xylulose-bound crystal structures of Escherichia coli XK have been determined and show a dimer composed of two domains separated by an open cleft. XK dimerization was observed directly by a cryo-EM reconstruction at 36 A resolution. Kinetic studies reveal that XK has a weak substrate-independent MgATP-hydrolyzing activity, and phosphorylates several sugars and polyols with low catalytic efficiency. Binding of pentulose and MgATP to form the reactive ternary complex is strongly synergistic. Although the steady-state kinetic mechanism of XK is formally random, a path is preferred in which D-xylulose binds before MgATP. Modelling of MgATP binding to XK and the accompanying conformational change suggests that sugar binding is accompanied by a dramatic hinge-bending movement that enhances interactions with MgATP, explaining the observed synergism. A catalytic mechanism is proposed and supported by relevant site-directed mutants. (c) 2006 Elsevier Ltd. All rights reserved.

2dx – User-friendly image processing for 2D crystals

B. Gipson; X. Zeng; Z. Y. Zhang; H. Stahlberg 

Electron crystallography determines the structure of two-dimensional (2D) membrane protein crystals and other 2D crystal systems. Cryotransmission electron microscopy records high-resolution electron micrographs, which require computer processing for three-dimensional structure reconstruction. We present anew software system 2dx, which is designed as a user-friendly, platform-independent software package for electron crystallography. 2dx assists in the management of an image-processing project, guides the user through the processing of 2D crystal images, and provides transparence for processing tasks and results. Algorithims are implemented in the form of script templates reminiscent of c-shell scripts. These templates can be easily modified or replaced by the user and can also execute modular stand-alone programs from the MRC software or from other image processing software packages. 2dx is available under the GNU General Public License at 2dx.org. (c) 2006 Elsevier Inc. All rights reserved.

Electron Crystallography of Membrane Proteins

Correction: Functional modulation of IFT kinesins extends the sensory repertoire of ciliated neurons in Caenorhabditis elegans

J. E. Evans; J. J. Snow; A. L. Gunnarson; G. Ou; H. Stahlberg et al. 

A homotetrameric kinesin-5, KLP61F, bundles microtubules and antagonizes Ncd in motility assays

L. Tao; A. Mogilner; G. Civelekogiu-Scholey; R. Wollman; J. Evans et al. 

Background: Mitosis depends upon the cooperative action of multiple microtubule (MT)-based motors. Among these, a kinesin-5, KLP61 F, and the kinesin-14, Ncd, are proposed to generate antagonistic-sliding forces that control the spacing of the spindle poles. We tested whether purified KLP61 F homotetramers and Ncd homodimers can generate a force balance capable of maintaining a constant spindle length in Drosophila embryos. Results: Using fluorescence microscopy and cryo-EM, we observed that purified full-length, motorless, and tailless KLP61F tetramers (containing a tetramerization domain) and Ncd dimers can all cross-link MTs into bundles in MgATP. In multiple-motor motility assays, KLP61 F and Ncd drive plus-end and minus-end MT sliding at 0.04 and 0.1 mu m/s, respectively, but the motility of either motor is decreased by increasing the mole fraction of the other. At the “balance point,” the mean velocity was zero and MTs paused briefly and then oscillated, taking similar to 0.3 mu m excursions at similar to 0.02 mu m/s toward the MT plus end and then the minus end. Conclusions: The results, combined with quantitative analysis, suggest that these motors could act as mutual brakes to modulate the rate of pole-pole separation and could maintain a prometaphase spindle displaying small fluctuations in its steady-state length.

Milestones in electron crystallography

L. Renault; H-T. Chou; P-L. Chiu; R. M. Hill; X. Zeng et al. 

Electron crystallography determines the structure of membrane embedded proteins in the two-dimensionally crystallized state by cryo-transmission electron microscopy imaging and computer structure reconstruction. Milestones on the path to the structure are high-level expression, purification of functional protein, reconstitution into two-dimensional lipid membrane crystals, high-resolution imaging, and structure determination by computer image processing. Here we review the current state of these methods. We also created an Internet information exchange platform for electron crystallography, where guidelines for imaging and data processing method are maintained. The server (http://2dx.org) provides the electron crystallography community with a central information exchange platform, which is structured in blog and Wiki form, allowing visitors to add comments or discussions. It currently offers a detailed step-by-step introduction to image processing with the MRC software program. The server is also a repository for the 2dx software package, a user-friendly image processing system for 2D membrane protein crystals.

Applications of Atomic Scale Scanning Transmission Electron Microscopy

N. Browning; R. Erni; C. Mitterbauer; L. Fu; M. Chi et al. 

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

Cryo-Electron Microscopy Analysis of a 105 kDa Protein Particle: The Xylulose Kinase from E. Coli

Chou; E. d. Luccio; D. Wilson; H. Stahlberg 

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

Avoiding Charge Induced Drift In Vitrified Biological Specimens Through Scanning Transmission Electron Microscopy

G. Nicotra; Chou; H. Stahlberg; N. Browning 

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2006

Double hexameric ring assembly of the type III protein translocase ATPase HrcN

S. A. Mueller; C. Pozidis; R. Stone; C. Meesters; M. Chami et al. 

The specialized type III secretion (T3S) apparatus of pathogenic and symbiotic Gram-negative bacteria comprises a complex transmembrane organelle and an ATPase homologous to the F-1-ATPase beta subunit. The T3S ATPase HrcN of Pseudomonas syringae associates with the inner membrane, and its ATP hydrolytic activity is stimulated by dodecamerization. The structure of dodecameric HrcN (HrcN(12)) determined to 1.6 nm by cryo-electron microscopy is presented. HrcN(12) comprises two hexameric rings that are probably stacked face-to-face by the association of their C-terminal domains. It is 11.5 +/- 1.0 nm in diameter, 12.0 +/- 2.0 nm high and has a 2.0-3.8 nm wide inner channel. This structure is compared to a homology model based on the structure of the F-1-beta-ATPase. A model for its incorporation within the T3S apparatus is presented.

Functional modulation of IFT kinesins extends the sensory repertoire of ciliated neurons in Caenorhabditis elegans (vol 172, pg 663, 2006)

J. E. Evans; J. J. Snow; A. L. Gunnarson; G. Ou; H. Stahlberg et al. 

Functional modulation of IFT kinesins extends the sensory repertoire of ciliated neurons in Caenorhabditis elegans

J. Evans; J. Snow; A. Gunnarson; G. Ou; H. Stahlberg et al. 

The diversity of sensory cilia on Coenorhabditis elegans neurons allows the animal to detect a variety of sensory stimuli. Sensory cilia are assembled by intraflagellar transport (IFT) kinesins, which transport ciliary precursors, bound to IFT particles, along the ciliary axoneme for Incorporation into ciliary structures. Using fluorescence microscopy of living animals and serial section electron microscopy of high pressure-frozen, freeze-substituted IFT motor mutants, we found that two IFT kinesins, homodimeric OSM-3 kinesin and heterotrimeric kinesin II, function in a partially redundant manner to build full-length amphid channel cilia but are completely redundant for building full-length amphid wing (AWC) cilia. This difference reflects cilia-specific differences in OSM-3 activity, which serves to extend distal singlets in channel cilia but not in AWC cilia, which lack such singlets. Moreover, AWC-specific chemotaxis assays reveal novel sensory functions for kinesin 11 in these wing cilia. We propose that kinesin II is a “canonical” IFT motor, whereas OSM-3 is an “accessory” IFT motor, and that subtle changes In the deployment or actions of these IFT kinesins can contribute to differences in cilia morphology, cilia function, and sensory perception.

The 4.5 angstrom structure of human AQP2

A. Schenk; P. Werten; S. Scheuring; B. de Groot; S. Muller et al. 

Located in the principal cells of the collecting duct, aquaporin-2 (AQP2) is responsible for the regulated water reabsorbtion in the kidney and is indispensable for the maintenance of body water balance. Disregulation or malfunctioning of AQP2 can lead to severe diseases such as nephrogenic diabetes insipidus, congestive heart failure, liver cirrhosis and preeclampsia. Here we present the crystallization of recombinantly expressed human AQP2 into two-dimensional protein-lipid arrays and their structural characterization by atomic force microscopy and electron crystallography. These crystals are double-layered sheets that have a diameter of up to 30 mu m, diffract to 3 angstrom(-1) and are stacked by contacts between their cytosolic surfaces. The structure determined to 4.5 angstrom resolution in the plane of the membrane reveals the typical aquaporin fold but also a particular structure between the stacked layers that is likely to be related to the cytosolic N and C termini. (c) 2005 Elsevier Ltd. All rights reserved.

Oligomeric structure of the Bacillus subtilis cell division protein DivIVA determined by transmission electron microscopy

H. Stahlberg; E. Kutejova; K. Muchova; M. Gregorini; A. Lustig et al. 

DivIVA from Bacillus subtilis is a bifunctional protein with distinct roles in cell division and sporulation. During vegetative growth, DivIVA regulates the activity of the MinCD complex, thus helping to direct cell division to the correct mid-cell position. DivIVA fulfils a quite different role during sporulation in B. subtilis when it directs the oriC region of the chromosome to the cell pole before asymmetric cell division. DivIVA is a 19.5 kDa protein with a large part of its structure predicted to form a tropomyosin-like alpha-helical coiled-coil. Here, we present a model for the quaternary structure of DivIVA, based on cryonegative stain transmission electron microscopy images. The purified protein appears as an elongated particle with lateral expansions at both ends producing a form that resembles a ‘doggy-bone’. The particle mass estimated from these images agrees with the value of 145 kDa measured by analytical ultracentrifugation suggesting 6- to 8-mers. These DivIVA oligomers serve as building blocks in the formation of higher order assemblies giving rise to strings, wires and, finally, two-dimensional lattices in a time-dependent manner.

Iplt-image processing library and toolkit for the electron microscopy community

A. Philippsen; A. Schenk; H. Stahlberg; A. Engel 

We present the foundation for establishing a modular, collaborative, integrated, open-source architecture for image processing of electron microscopy images, named iplt. It is designed around object oriented paradigms and implemented using the programming languages C++ and Python. In many aspects it deviates from classical image processing approaches. This paper intends to motivate developers within the community to participate in this on-going project. The iplt homepage can be found at http: www.iplt.org. (C) 2003 Elsevier Inc. All rights reserved.

Type III protein translocase – Hrcn is a peripheral membrane ATPase that is activated by oligomerization

C. Pozidis; A. Chalkiadaki; A. Gomez-Serrano; H. Stahlberg; I. Brown et al. 

Type III protein secretion (TTS) is catalyzed by translocases that span both membranes of Gram-negative bacteria. A hydrophilic TTS component homologous to F-1/V-1-ATPases is ubiquitous and essential for secretion. We show that hrcN encodes the putative TTS ATPase of Pseudomonas syringae pathovar phaseolicola and that HrcN is a peripheral protein that assembles in clusters at the membrane. A decahistidinyl HrcN derivative was overexpressed in Escherichia coli and purified to homogeneity in a folded state. Hydrodynamic analysis, cross-linking, and electron microscopy revealed four distinct HrcN forms: I, 48 kDa ( monomer); II, similar to300 kDa ( putative hexamer); III, 575 kDa ( dodecamer); and IV, similar to3.5 MDa. Form III is the predominant form of HrcN at the membrane, and its ATPase activity is dramatically stimulated (> 700-fold) over the basal activity of Form I. We propose that TTS ATPases catalyze protein translocation as activated homo-oligomers at the plasma membrane.

Assessing the structure of membrane proteins: combining different methods gives the full picture

H. Stahlberg; A. Engel; A. Philippsen 

The rotor stoichiometry of F-ATPases has been revealed by the combined approaches of X-ray diffraction (XRD), electron crystallography, and atomic force microscopy (AFM). XRD showed the rotor from the yeast mitochondrial F-ATPase to contain 10 subunits. AFM was used to visualize the tetradecameric chloroplast rotors, and electron crystallography and AFM together revealed the rotors from Ilyobacter tartaricus to be composed of 11 subunits. While biochemical methods had determined an approximate stoichiometric value, precise measurements and new insights into a species-dependent rotor stoichiometry became available by applying the three structural tools together. The structures of AQP1, a water channel, and GlpF, a glycerol channel, were determined by electron crystallography and XRD. The combination of both of these structural tools with molecular dynamics simulations gave a differentiated description of the mechanisms determining the selectivity of water and glycerol channels. This illustrates that the combination of different methods in structural biology reveals more than each method alone.

Progress in the analysis of membrane protein structure and function

P. Werten; H. Remigy; B. de Groot; D. Fotiadis; A. Philippsen et al. 

Structural information on membrane proteins is sparse, yet they represent an important class of proteins that is encoded by about 30% of all genes. Progress has primarily been achieved with bacterial proteins, but efforts to solve the structure of eukaryotic membrane proteins are also increasing. Most of the structures currently available have been obtained by exploiting the power of X-ray crystallography. Recent results, however, have demonstrated the accuracy of electron crystallography and the imaging power of the atomic force microscope. These instruments allow membrane proteins to be studied while embedded in the bi-layer, and thus in a functional state. The low signal-to-noise ratio of cryo-electron microscopy is overcome by crystallizing membrane proteins in a two-dimensional protein-lipid membrane, allowing its atomic structure to be determined. In contrast, the high signal-to-noise ratio of atomic force microscopy allows individual protein surfaces to be imaged at subnanometer resolution, and their conformational states to be sampled. This review summarizes the steps in membrane protein structure determination and illuminates recent progress. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Sampling the conformational space of membrane protein surfaces with the AFM

S. Scheuring; D. Muller; H. Stahlberg; H. Engel; A. Engel 

The atomic force microscope acquires topographs of single native membrane proteins at subnanometer resolution. Owing to the high signal-to-noise ratio, such images allow the conformational space of membrane protein surfaces to be sampled. This is demonstrated by topographs of porin OrnpF, aquaporin-Z, and bacteriorhodopsin, all recorded at a lateral resolution of <7 &ANGS; and a vertical resolution of &SIM;1 &ANGS;. The amplitudes of the domain movements were estimated from a large number of single molecule topographs and the corresponding energy landscapes calculated. To visualize the motion of protein domains, movies were generated by similarity ranking of the observed protein configurations. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00249-001-0197-8.

Charting and unzipping the surface layer of Corynebacterium glutamicum with the atomic force microscope

S. Scheuring; H. Stahlberg; M. Chami; C. Houssin; J. Rigaud et al. 

Bacterial surface layers (S-layers) are extracellular protein networks that act as molecular sieves and protect a large variety of archaea and bacteria from hostile environments. Atomic force microscopy (AFM) was used to asses the S-layer of Corynebacterium glutamicum formed of PS2 proteins that assemble into hexameric complexes within a hexagonal lattice. Native and trypsin-treated S-layers were studied. Using the AFM stylus as a nanodissector, native arrays that adsorbed to mica as double layers were separated. All surfaces of native and protease-digested S-layers were imaged at better than 1 nm lateral resolution. Difference maps of the topographies of native and proteolysed samples revealed the location of the cleaved C-terminal fragment and the sidedness of the S-layer. Because the corrugation depths determined from images of both sides span the total thickness of the S-layer, a three-dimensional reconstruction of the S-layer could be calculated. Lattice defects visualized at 1 nm resolution revealed the molecular boundaries of PS2 proteins. The combination of AFM imaging and single molecule force spectroscopy allowed the mechanical properties of the Corynebacterium glutamicum S-layer to be examined. The results provide a basis for understanding the amazing stability of this protective bacterial surface coat.

Aquaglyceroporins: Channel proteins with a conserved core, multiple functions, and variable surfaces

Two-dimensional crystals: a powerful approach to assess structure, function and dynamics of membrane proteins

H. Stahlberg; D. Fotiadis; S. Scheuring; H. Remigy; T. Braun et al. 

Electron crystallography and atomic force microscopy allow the study of two-dimensional membrane protein crystals. While electron crystallography provides atomic scale three-dimensional density maps, atomic force microscopy gives insight into the surface structure and dynamics at sub-nanometer resolution. Importantly, the membrane protein studied is in its native environment and its function can be assessed directly. The approach allows both the atomic structure of the membrane protein and the dynamics of its surface to be analyzed. In this way, the function-related conformational changes can be assessed, thus providing a detailed insight on the molecular mechanisms of essential biological processes. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

ATP synthase: constrained stoichiometry of the transmembrane rotor

D. Muller; N. Dencher; T. Meier; P. Dimroth; K. Suda et al. 

Recent structural data suggest that the number of identical subunits (c or III) assembled into the cation-powered rotor of F1F0 ATP synthase depends on the biological origin. Atomic force microscopy allowed individual subunits of the cylindrical transmembrane rotors from spinach chloroplast and from Ilyobacter tartaricus ATP synthase to be directly visualized in their native-like environment. Occasionally, individual rotors exhibit structural gaps of the size of one or more subunits. Complete rotors and arch-shaped fragments of incomplete rotors revealed the same diameter within one ATP synthase species. These results suggest the rotor diameter and stoichiometry to be determined by the shape of the subunits and their nearest neighbor interactions. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Bacterial Na+-ATP synthase has an undecameric rotor

H. Stahlberg; D. Muller; K. Suda; D. Fotiadis; A. Engel et al. 

Synthesis of adenosine triphosphate (ATP) by the F1F0 ATP synthase involves a membrane-embedded rotary engine, the F-0 domain, which drives the extra-membranous catalytic F-1 domain. The F-0 domain consists of subunits a(1)b(2) and a cylindrical rotor assembled from 9-14 alpha -helical hairpin-shaped c-subunits. According to structural analyses, rotors contain 10 c-subunits in yeast and 14 in chloroplast ATP synthases. We determined the rotor stoichiometry of Ilyobacter tartaricus ATP synthase by atomic force microscopy and cryo-electron microscopy, and show the cylindrical sodium-driven rotor to comprise 11 c-subunits.

Electron Cryomicroscopy

The aquaporin superfamily: Structure and function

The 6.9-angstrom structure of GlpF: A basis for homology modeling of the glycerol channel from Escherichia coli

H. Stahlberg; T. Braun; B. de Groot; A. Philippsen; M. Borgnia et al. 

The three-dimensional structure of GlpF, the glycerol facilitator of Escherichia coli, was determined by cryo-electron microscopy, The 6.9-Angstrom density map calculated from images of two-dimensional crystals shows the GlpF helices to be similar to those of AQP1, the erythrocyte water channel, While the helix arrangement of GlpF does not reflect the larger pore diameter as seen in the projection map, additional peripheral densities observed in GlpF are compatible with the 31 additional residues in loops C and E, which accordingly do not interfere with the inner channel construction. Therefore, the atomic structure of AQP1 was used as a basis for homology modeling of the GlpF channel, which is predicted to be free of bends, wider, and more vertically oriented than the AQP1 channel. Furthermore, the residues facing the GlpF channel exhibit an amphiphilic nature, being hydrophobic on one side and hydrophilic on the other side. This property may partially explain the contradiction of glycerol diffusion but limited water permeation capacity. (C) 2000 Academic Press.

The 3.7 angstrom projection map of the glycerol facilitator GlpF: a variant of the aquaporin tetramer

T. Braun; A. Philippsen; S. Wirtz; M. Borgnia; P. Agre et al. 

GlpF, the glycerol facilitator protein of Escherichia coli, is an archetypal member of the aquaporin superfamily. To assess its structure, recombinant histidine-tagged protein was overexpressed, solubilized in octylglucoside and purified to homogeneity. Negative stain electron microscopy of solubilized GlpF protein revealed a tetrameric structure of similar to 80 Angstrom side length. Scanning transmission electron microscopy yielded a mass of 170 kDa, corroborating the tetrameric nature of GlpF. Reconstitution of GlpF in the presence of lipids produced highly ordered two-dimensional crystals, which diffracted electrons to 3.6 Angstrom resolution. Cryoelectron microscopy provided a 3.7 Angstrom projection map exhibiting a unit cell comprised of two tetramers. In projection, GlpF is similar to AQP1, the erythrocyte water channel. However, the major density minimum within each monomer is distinctly larger in GlpF than in AQP1.

Surface tongue-and-groove contours on lens MIP facilitate cell-to-cell adherence

D. Fotiadis; L. Hasler; D. Muller; H. Stahlberg; J. Kistler et al. 

The lens major intrinsic protein (MTP, AQP0) is known to function as a water and solute channel. However, MIP has also been reported to occur in close membrane contacts between lens fiber cells, indicating that it has adhesive properties in addition to its channel function. Using atomic force and cryo-electron microscopy we document that crystalline sheets reconstituted from purified ovine lens MIP mostly consisted of two layers. MIP lattices in the apposing membranes were in precise register, and determination of the membrane sidedness demonstrated that MIP molecules bound to each other via their extracellular surfaces. The surface structure of the latter was resolved to 0.61 nm and revealed two protruding domains providing a tight “tongue-and-groove” fit between apposing MIP molecules. Cryo-electron crystallography produced a projection map at 0.69 nm resolution with a mirror symmetry axis at 45 degrees to the lattice which was consistent with the double-layered nature of the reconstituted sheets. These data strongly suggest an adhesive function of MIP, and strengthen the view that MIP serves dual roles in the lens. (C) 2000 Academic Press.

The aquaporin sidedness revisited

S. Scheuring; P. Tittmann; H. Stahlberg; P. Ringler; M. Borgnia et al. 

Aquaporins are transmembrane water channel proteins, which play important functions in the osmoregulation and water balance of microorganisms, plants, and animal tissues. All aquaporins studied to date are thought to be tetrameric assemblies of four subunits each containing its own aqueous pore. Moreover, the subunits contain an internal sequence repeat forming two obversely symmetric hemichannels predicted to resemble an hour-glass. This unique arrangement of two highly related protein domains oriented at 180 degrees to each other poses a significant challenge in the determination of sidedness. Aquaporin Z (AqpZ) from Escherichia coli was reconstituted into highly ordered two-dimensional crystals. They were freeze-dried and metal-shadowed to establish the relationship between surface structure and underlying protein density by electron microscopy. The shadowing of some surfaces was prevented by protruding aggregates. Thus, images collected from freeze-dried crystals that exhibited both metal-coated and uncoated regions allowed surface relief reconstructions and projection maps to be obtained from the same crystal. Cross-correlation peak searches along lattices crossing metal-coated and uncoated regions allowed an unambiguous alignment of the surface reliefs to the underlying density maps. AqpZ topographs previously determined by AFM could then be aligned with projection maps of AqpZ, and finally with human erythrocyte aquaporin-1 (AQP1). Thereby features of the AqpZ topography could be interpreted by direct comparison to the 6 Angstrom three-dimensional structure of AQP1. We conclude that the sidedness we originally proposed for aquaporin density maps was inverted. (C) 2000 Academic Press.

Proton-powered turbine of a plant motor

H. Seelert; A. Poetsch; N. A. Dencher; A. Engel; H. Stahlberg et al. 

Domain structure of secretin PulD revealed by limited proteolysis and electron microscopy

N. Nouwen; H. Stahlberg; A. Pugsley; A. Engel 

Secretins, a superfamily of multimeric outer membrane proteins, mediate the transport of large macromolecules across the outer membrane of Gramnegative bacteria. Limited proteolysis of secretin PulD from the Klebsiella oxytoca pullulanase secretion pathway showed that it consists of an N-terminal domain and a protease-resistant C-terminal domain that remains multimeric after proteolysis. The stable C-terminal domain starts just before the region in PulD that is highly conserved in the secretin superfamily and apparently lacks the region at the C-terminal end to which the secretin-specific pilot protein PulS binds. Electron microscopy showed that the stable fragment produced by proteolysis is composed of two stacked rings that encircle a central channel and that it lacks the peripheral radial spokes that are seen in the native complex. Moreover, the electron microscopic images suggest that the N-terminal domain folds back into the large cavity of the channel that is formed by the C-terminal domain of the native complex, thereby occluding the channel, consistent with previous electrophysiological studies showing that the channel is normally closed.

High resolution AFM topographs of the Escherichia coli water channel aquaporin Z

S. Scheuring; P. Ringler; M. Borgnia; H. Stahlberg; D. Muller et al. 

Aquaporins form a large family of membrane channels involved in osmoregulation, Electron crystallography has shown monomers to consist of six membrane spanning a-helices confirming sequence based predictions. Surface exposed loops are the least conserved regions, allowing differentiation of aquaporins. Atomic force microscopy was used to image the surface of aquaporin Z, the water channel of Escherichia coli, Recombinant protein with an N-terminal fragment including 10 histidines was isolated as a tetramer by Ni-affinity chromatography, and reconstituted into two-dimensional crystals with p42(1)2 symmetry. Small crystalline areas with p4 symmetry were found as well, Imaging both crystal types before and after cleavage of the N-termini allowed the cytoplasmic surface to be identified; a drastic change of the cytoplasmic surface accompanied proteolytic cleavage, while the extracellular surface morphology did not change. Flexibility mapping and volume calculations identified the longest loop at the extracellular surface. This loop exhibited a reversible force-induced conformational change.

Structure of the water channel AqpZ from Escherichia coli revealed by electron crystallography

P. Ringler; M. Borgnia; H. Stahlberg; P. Maloney; P. Agre et al. 

Molecular water channels (aquaporins) allow living cells to adapt to osmotic variations by rapid and specific diffusion of water molecules. Aquaporins are present in animals, plants, algae, fungi and bacteria. Here we present an electron microscopic analysis of the most ancient water channel described so far: the aquaporin Z (AqpZ) of Escherichia coli. A recombinant AqpZ with a poly(histidine) tag at the N terminus has been constructed, overexpressed and purified to homogeneity. Solubilized with octylglucoside, the purified AqpZ remains associated as a homotetramer, and assembles into highly ordered two-dimensional tetragonal crystals with unit cell dimensions a=b=95 Angstrom, gamma=90 degrees when reconstituted by dialysis in the presence of Lipids. Three-dimensional reconstruction of negatively stained lattices revealed the p42(1)2 packing arrangement that is also observed with the human erythrocyte water channel (AQP1). The 8 Angstrom projection map of the AqpZ tetramer in frozen hydrated samples is similar to that of AQP1, consistent with the high sequence homology between these proteins. (C) 1999 Academic Press.

Mitochondrial Lon of Saccharomyces cerevisiae is a ring-shaped protease with seven flexible subunits

H. Stahlberg; E. Kutejova; K. Suda; B. Wolpensinger; A. Lustig et al. 

Lon (or La) is a soluble, homooligomeric ATP-dependent protease. Mass determination and cryoelectron microscopy of pure mitochondrial Lon from Saccharomyces cerevisiae identify Lon as a flexible ring-shaped heptamer. In the presence of ATP or 5′-adenylylimidodiphosphate, most of the rings are symmetric and resemble other ATP-driven machines that mediate folding and degradation of proteins. In the absence of nucleotides, most of the rings are distorted, with two adjacent subunits forming leg-like protrusions. These results suggest that asymmetric conformational changes serve to power processive unfolding and translocation of substrates to the active site of the Lon protease.

The reaction center complex from the green sulfur bacterium Chlorobium tepidum: A structural analysis by scanning transmission electron microscopy

H. Remigy; H. Stahlberg; D. Fotiadis; S. Muller; B. Wolpensinger et al. 

The three-dimensional (3D) structure of the reaction center (RC) complex isolated from the green sulfur bacterium Chlorobium tepidum was determined from projections of negatively stained preparations by angular reconstitution. The purified complex contained the PscA, PscC, PscB, PscD subunits and the Fenna-Matthews-Olson (FMO) protein. Its mass was found to be 454 kDa by scanning transmission electron microscopy (STEM), indicating the presence of two copies of the PscA subunit, one copy of the PscB and PscD subunits, three FMO proteins and at least one copy of the PscC subunit. An additional mass peak at 183 kDa suggested that FMO trimers copurify with the RC complexes. Images of negatively stained RC complexes were recorded by STEM and aligned and classified by multivariate statistical analysis. Averages of the major classes indicated that different morphologies of the elongated particles (length = 19 nm, width = 8 nm) resulted from a rotation around the long axis. The 3D map reconstructed from these projections allowed visualization of the RC complex associated with one FMO trimer. A second FMO trimer could be correspondingly accommodated to yield a symmetric complex, a structure observed in a small number of side views and proposed to be the intact form of the RC complex. (C) 1999 Academic Press.

Friction anisotropy and asymmetry of a compliant monolayer induced by a small molecular tilt

M. Liley; D. Gourdon; D. Stamou; U. Meseth; T. Fischer et al. 

Lateral force microscopy in the wearless regime was used to study the friction behavior of a lipid monolayer on mica. In the monolayer, condensed domains with long-range orientational order of the lipid molecules were present. The domains revealed unexpectedly strong friction anisotropies and non-negligible friction asymmetries, The angular dependency of these effects correlated well with the tilt direction of the alkyl chains of the monolayer, as determined by electron diffraction and Brewster angle microscopy, The molecular tilt causing these frictional effects was less than 15 degrees, demonstrating that even small molecular tilts can make a major contribution to friction.

Are the light-harvesting I complexes from Rhodospirillum rubrum arranged around the reaction center in a square geometry?

H. Stahlberg; J. Dubochet; H. Vogel; R. Ghosh 

The basic photosynthetic unit contg. the reaction center and the light-harvesting I complex (RC-LHI) of the purple non-sulfur bacterium Rhodospirillum rubrum was purified and reconstituted into two-dimensional (2D) membrane crystals. Transmission electron microscopy using conventional techniques and cryoelectron microscopy of the purified single particles and of 2D crystals yielded a projection of the RC-LHI complex at a resoln. of at least 1.6 nm. In this projection the LHI ring appears to have a square symmetry and packs in a square crystal lattice. The square geometry of the LHI ring was obsd. also in images of single isolated particles of the RC-LHI complex. However, although the LHI units are packed identically within the crystal lattice, a new rotational anal. developed here showed that the reaction centers take up one of four possible orientations within the ring. This fourfold disorder supports our interpretation of a square ring symmetry and suggests that a hitherto undetected component may be present within the photosynthetic unit. (c) 1998 Academic Press. [on SciFinder (R)]

The reaction center of the photounit of Rhodospirillum rubrum is anchored to the light-harvesting complex with four-fold rotational disorder

H. Stahlberg; J. Dubochet; H. Vogel; R. Ghosh 

The minimal photounit of the photosynthetic membranes of the purple non-sulfur bacterium Rhodospirillum rubrum, comprising the reaction center and the light-harvesting complex has been purified and crystd. in two dimensions in the presence of added phospholipids, and subsequently visualized by electron microscopy after neg.-staining. The position of the reaction centers within the light-harvesting ring has been detd. at low resoln. by the application of a new anal. for rotationally disordered identical units (here the reaction centers) within a two-dimensional cryst. lattice comprised of perfectly aligned unit cells (here the light-harvesting complexes). The reaction center was found to preferentially occupy one of four orientations within the light-harvesting complex. The light-harvesting complex appears to be distored to C4 symmetry,thus assuming a squarish shape when visualized by neg. staining. A tentative structural model of the reaction center-light-harvesting complex photounit which fits the exptl. data is proposed. [on SciFinder (R)]

Chloroplast F0F1 ATP synthase imaged by atomic force microscopy

D. Neff; S. Tripathi; K. Middendorf; H. Stahlberg; H. Butt et al. 

The F0F1 ATP synthase of chloroplasts was imaged using atomic force microscopy (AFM) in contact mode under physiological conditions. Chloroplast (CF0F1) ATP synthases were reconstituted into liposomes, Liposomes were adsorbed on a mica surface where they spread and formed lipid bilayers containing CF0F1 ATP synthases which could be imaged. From these reconstituted CF0F1 ATP synthases, the CF1 part could be removed either by application of a chemical denaturant or less efficiently by mechanical stripping with the AFM tip. Embedded in the lipid bilayer were seen ring-like structures with a central dimple with outer diameters of 20 +/- 3 nm (chemical denaturant) and ca, 7 nm (mechanical stripping), respectively, Ring-like structures were also observed in a protein-free lipid bilayer, These had diameters of 30 +/- 5 nm and could be clearly distinguished from the structures observed after mechanical stripping, Hence, the ring-like structures observed after mechanical stripping might represent the intrinsic membrane domain CF0 or the oligomer of its subunit III, In addition, isolated CF1 adsorbed directly onto the mica surface was imaged, In accordance with the size known from electron microscopy, a diameter of 13 +/- 4 nm was measured, (C) 1997 Academic Press.

Sulfur-bearing lipids for the covalent attachment of supported lipid bilayers to gold surfaces: a detailed characterization and analysis

C. Duschl; M. Liley; H. Lang; A. Ghandi; S. M. Zakeeruddin et al. 

In a recent paper, a new class of synthetic lipids, which self-assemble to form monolayers on gold surfaces, was introduced. The addn. of a monolayer of phospholipids to these layers results in robust supported lipid bilayers designed to reconstitute transmembrane proteins. This paper presents a detailed characterization of the monolayers and bilayers formed by these lipids and assesses their ability for the incorporation of membrane-spanning proteins. Film balance methods, electron microscopy, surface plasmon resonance, Fourier transform IR measurements and Raman spectroscopy show the feasibility of this concept. However, further modifications to improve the thiolipids are necessary. A new strategy for the formation of laterally structured supported lipid bilayers is discussed and initial results are presented. [on SciFinder (R)]