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.
Highlights
The concept of structural polymorphism has been associated with structure research in the beginning
We choose a few milestone cases of cryo-electron microscope (cryo-EM) structures including sub-100 kDa proteins, membrane proteins, and protein machineries to illustrate the exciting frontier of using cryo-EM to visualize structural polymorphism of protein complexes
Structural polymorphism was best approached by crystal studies, but the physiological relevance of those structures were often in question due to the potential artifact induced by crystal packing
Summary
The concept of structural polymorphism has been associated with structure research in the beginning. Those observations were consistent with those movements observed in the crystal structures (Shibayama et al, 2014) The latter cryo-EM study of haemoglobin with thorough classification analyses clearly demonstrates that high-resolution single-particle cryo-EM is applicable to resolve distinct, biologically relevant conformational states of a sub-100 kDa complex. As raising temperature can increase protein dynamics and 4°C is much far away from the point where most of enzymes are catalytically active, it is likely that the structures obtained from those cryo-EM studies may not closely reflect those of the macromolecules in action To address this issue Chang et al (Chang et al, 2021) modified the Vitrobot freezing device to enable the investigation of the structure of ketol-acid reductoisomerase (KARI) found in a hot spring bacteria (Sulfolobus acidocaldarius) that exhibits optimal enzyme activity in the range of 60–70°C (Chen et al, 2018). A recent study of TRPV1 at elevated temperature has revealed the snapshots of heat-dependent opening of TRPV1 in the presence of capsaicin that underlies the mechanism of nociception (Kwon et al, 2021)
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