Proteins are dynamic systems whose free energy surface determines their motions and characteristic timescales. Backbone motions and side-chain rotations involve transitions between minima separated by small barriers and typically occur on picosecond to nanosecond timescales. The dynamics associated with the activity of a protein frequently involve large-amplitude motions of entire domains that occur on a timescale of microseconds to milliseconds. Unfortunately, such fast dynamics cannot be studied with conventional cryo-electron microscopy (cryo-EM) approaches.
Our group has developed a novel time-resolved cryo-EM technique capable of probing protein dynamics on the microsecond timescale. Our approach involves rapidly melting a cryo specimen in situ with a laser beam. The sample remains liquid for the duration of the laser pulse, offering a tunable time window in which the dynamics of the embedded particles can be induced in their native liquid environment. After the laser pulse, the sample revitrifies in just a few microseconds, trapping particles in their transient configurations, so that they can subsequently be characterized with existing cryo-EM imaging techniques.
Related publications:
Fast Viral Dynamics Revealed by Microsecond Time-Resolved Cryo-EM
Oliver. F. Harder, Sarah V. Barrass, Marcel Drabbels, Ulrich J. Lorenz. Nat. Commun. 14, 5649 (2023)
Near-atomic resolution reconstructions from in situ revitrified cryo sample
Fast Viral Dynamics Revealed by Microsecond Time-Resolved Cryo-EM
Oliver. F. Harder, Sarah V. Barrass, Marcel Drabbels, Ulrich J. Lorenz.
Microsecond melting and revitrification of cryo samples with a correlative light-electron microscopy approach G. Bongiovanni, O.F. Harder, M. Drabbels, U.J. Lorenz. Frontiers 9, 1044509 (2022)
Microsecond Melting and Revitrification of Cryo Samples: Protein Structure and Beam-Induced Motion. O.F. Harder, J.M. Voss, P.K. Olshin, M. Drabbels, U.J. Lorenz. Acta Cryst D. 78, 883–889 (2022)
Microsecond Melting and Revitrification of Cryo Samples. J.M. Voss, O.F. Harder, P.K. Olshin, M. Drabbels, U.J. Lorenz. Struct. Dyn. (Editor’s Pick) 8, 054302 (2021)
Rapid Melting and Revitrification as an Approach to Microsecond Time-Resolved Cryo-Electron Microscopy. J.M. Voss, O.F. Harder, P.K. Olshin, M. Drabbels, U.J. Lorenz. Chem. Phys. Lett. (selected Frontiers Article) 778, 138812 (2021)