The physical origins of specimen movement in electron cryomicroscopy and how to eliminate it
Electron cryomicroscopy (cryoEM) is an important imaging technique for determining the atomic structures of biological macromolecules and complexes. Success in determining a structure by cryoEM depends on being able to prepare a thin frozen specimen of the molecules of interest on a small metal support, called a grid. Most high-resolution information loss in cryomicrographs stems from (1) radiation damage, and (2) particle movement in the vitrified specimen during imaging. This movement has been an unexplained problem for the field ever since Jaques Dubochet invented the method, more than three decades ago. In this work, experiments were performed to elucidate the physical causes of this movement and to formulate a theory that describes it. The movement is caused by buckling during freezing and subsequent deformation during imaging of the suspended ice within the holes of the grid. Surprisingly, there exists a critical threshold for this phenomenon, that depends directly on the shape of the frozen water layer. Below this threshold (width:thickness ratio for a given geometry), specimen movement is eliminated. Based on these findings, a simple solution to the movement problem in single-particle cryoEM is proposed: a new all-gold specimen support, HexAuFoil, where the foil hole diameter is tuned to be below the threshold, only 10 times larger than the specimen thickness (in contrast to the typical factor of 100 in commercially available supports). Movement-free imaging increases the information content of cryoEM movies, increases the throughput of current microscopes, and reduces the computational demands on processing them.
Movement-free imaging also allows for a new approach to cryoEM reconstruction from 2D images: zero-dose extrapolation. A program that uses a radiation damage model to calculate the 3D structure of the undamaged molecule, before the onset of radiation damage, from movement-free data is developed. In collaboration with colleagues, we used HexAuFoil specimen supports and this approach to determine the structures of three protein complexes: (1) the SARS-CoV-2 RNA-dependent RNA polymerase in presence of RNA and the inhibitor favipiravir, (2) the light-harvesting 2 antenna complex of the purple bacterium Mch. purpuratum, and (3) the double-ring photosystem of G. phototrophica. These examples illustrate how movement-free imaging allowed us to better visualise ligand binding, metal coordination, solvent molecules, alternative side chain conformations, lipid bilayers and other important structural features of the molecules.
This work also addresses a major practical bottleneck in modern cryoEM: the availability of high quality grids. Starting from the HexAuFoil design, the first integrated, wafer-scale method for manufacturing specimen supports by the thousand is developed. This method is based on techniques from the semiconductor industry. In contrast to current manufacturing methods, it does not require handling of individual grids, allowing large batches of various types of specimen supports to be produced in a scaleable manner with little labor.
Understanding how and why plunge frozen vitreous specimens move unlocks the potential for further improvements in cryoEM. For example, movement-free imaging on a HexAuFoil support at 13 Kelvin is now demonstrated, meaning that we may soon be able to take advantage of the reduced secondary effects of radiation damage in cryoEM at lower temperature.