Cryo-EM Analyses Permit Visualization of Structural Polymorphism of Biological Macromolecules

Abstract

The functions of biological macromolecules are often associated with conformational malleability of the structures. This phenomenon of chemically identical molecules with different structures is coined structural polymorphism. Conventionally, structural polymorphism is observed directly by structural determination at the density map level from X-ray crystal diffraction. Although crystallography approach can report the conformation of a macromolecule with the position of each atom accurately defined in it, the exploration of structural polymorphism and interpreting biological function in terms of crystal structures is largely constrained by the crystal packing. An alternative approach to studying the macromolecule of interest in solution is thus desirable. With the advancement of instrumentation and computational methods for image analysis and reconstruction, cryo-electron microscope (cryo-EM) has been transformed to be able to produce “in solution” structures of macromolecules routinely with resolutions comparable to crystallography but without the need of crystals. Since the sample preparation of single-particle cryo-EM allows for all forms co-existing in solution to be simultaneously frozen, the image data contain rich information as to structural polymorphism. The ensemble of structure information can be subsequently disentangled through three-dimensional (3D) classification analyses. In this review, we highlight important examples of protein structural polymorphism in relation to allostery, subunit cooperativity and function plasticity recently revealed by cryo-EM analyses, and review recent developments in 3D classification algorithms including neural network/deep learning approaches that would enable cryo-EM analyese in this regard. Finally, we brief the frontier of cryo-EM structure determination of RNA molecules where resolving the structural polymorphism is at dawn.

Use of phase plate cryo-EM reveals conformation diversity of therapeutic IgG with 50 kDa Fab fragment resolved below 6 Å

“…Compared to cryo-ET, single-particle approach is an averaging technique that it is less sensitive to structural variation. Nonetheless, companion structural variation analysis allows capturing structural polymorphism in hydrated state [12,39], even at medium resolutions. Given that IgG is a flexible protein composed of small domains (50 kDa) and thus considered as an unfavorable target for high-resolution cryo-EM analysis, we aimed to apply single-particle cryo-EM to Rituximab, a therapeutic IgG, to aim for medium-resolution information for exploring its solution conformations.…”

Cryo-EM inspired NMR analysis reveals a pH-induced conformational switching mechanism for imparting dynamics to Betanodavirus protrusions

“…Compared to X-ray crystallography, cryo-EM represents an alternative high-resolution technique with the advantages of providing easier access to close-to-native state structures in conditions mimicking native environments and allowing for co-existing conformations to be disentangled [22]. Those advantages are crucial for capturing snapshots of dynamic structures or structural alterations induced by changes in pH, ions [23], or temperature [24].…”

Use of phase plate cryo-EM reveals conformation diversity of therapeutic IgG with 50 kDa Fab fragment resolved below 6 Å

“…This question could be better addressed using techniques that do not constrain antibody conformations in crystals. Compared to X-ray crystallography, cryo-electron microscopy (cryo-EM) is virtually a solution technique capable of accessing structural polymorphism in solution because the recorded images represent a body of mixed data of all co-existing solution conformations 12 . In addition, cryo-EM imaging method is versatile in that it accommodates nearly all sample conditions in response to physicochemical or biochemical manipulations.…”