X-rays in the Cryo-Electron Microscopy Era: Structural Biology’s Dynamic Future

Abstract

Over the past several years, single-particle cryo-electron microscopy (cryo-EM) has emerged as a leading method for elucidating macromolecular structures at near-atomic resolution, rivaling even the established technique of X-ray crystallography. Cryo-EM is now able to probe proteins as small as hemoglobin (64 kDa), while avoiding the crystallization bottleneck entirely. The remarkable success of cryo-EM has called into question the continuing relevance of X-ray methods, particularly crystallography. To say that the future of structural biology is either cryo-EM or crystallography, however, would be misguided. Crystallography remains better suited to yield precise atomic coordinates of macromolecules under a few hundred kDa in size, while the ability to probe larger, potentially more disordered assemblies is a distinct advantage of cryo-EM. Likewise, crystallography is better equipped to provide high-resolution dynamic information as a function of time, temperature, pressure, and other perturbations, whereas cryo-EM offers increasing insight into conformational and energy landscapes, particularly as algorithms to deconvolute conformational heterogeneity become more advanced. Ultimately, the future of both techniques depends on how their individual strengths are utilized to tackle questions on the frontiers of structural biology. Structure determination is just one piece of a much larger puzzle: a central challenge of modern structural biology is to relate structural information to biological function. In this perspective, we share insight from several leaders in the field and examine the unique and complementary ways in which X-ray methods and cryo-EM can shape the future of structural biology.

Experimentally determined long intrinsically disordered protein regions are now abundant in the Protein Data Bank

“…Indeed, Pearson's correlation coefficients are 0.17 and -0.09 for X-ray and cryo-EM, respectively. It is important to mention that the way in which these techniques determine the resolution is different [23], and consequently their maps and models could not be equivalent [24]. In our data set, solely 2% of structures were obtained by cryo-EM.…”

Bringing diffuse X-ray scattering into focus

“…As the structural biology toolkit expands, X-ray scattering, including diffuse scattering, still provides unique capabilities to probe conformational ensembles over many length scales, as captured in the recent review by Meisburger et al [ 26 •• ]. Ultimately, the better models of concerted motions will have far ranging impact beyond the average structure that is accessible using conventional X-ray crystallography and cryo-electron microscopy data, yielding a deeper understanding of biochemical mechanism [ 68 ].…”

Bringing Diffuse X-ray Scattering Into Focus

“…As the structural biology toolkit expands, X-ray scattering, including diffuse scattering, still provides unique capabilities to probe conformational ensembles over many length scales, as captured in the recent review by Meisburger et al [26]. Ultimately, the better models of concerted motions will have far ranging impact beyond the average structure that is accessible using conventional X-ray crystallography and cryo-electron microscopy data, yielding a deeper understanding of biochemical mechanism [68]. Highlighted References:…”