The number of structures released yearly in the PDB is shown by technique: X-ray crystallography (yellow), NMR (orange), and EM (blue). Historical milestones in macromolecular X-ray crystallography and electron microscopy. Finally, we discuss how the two techniques might leverage their unique capabilities to shape the future of structural biology, both separately and in parallel. In the following sections, we share insights from leaders in both fields and describe four key considerations in envisioning the future of the two techniques: crystallization, resolution and model quality, temperature, and dynamics. Will cryo-EM surpass X-ray crystallography? To provide context for this question, we first review the intertwined history of X-ray crystallography and EM. However, in the last five years or so, cryo-EM has experienced a “resolution revolution,” resulting in a flurry of high-resolution structures, 1 and at the time of this writing (October, 2017), has surpassed NMR in the number of structures released in the PDB per year ( Figure 1). Electron microscopy (EM), on the other hand, is responsible for just over 1,200 protein structures. Nuclear magnetic resonance (NMR) spectroscopy comes in second, claiming responsibility for 10,500 protein structures. Since its inception, X-ray crystallography has been used to determine over 112,000 structures of proteins in the Protein Data Bank (PDB), making it the most widely used technique for protein structure determination. 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. 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. Ultimately, the future of both techniques depends on how their individual strengths are utilized to tackle questions on the frontiers of structural biology. 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. 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. To say that the future of structural biology is either cryo-EM or crystallography, however, would be misguided. The remarkable success of cryo-EM has called into question the continuing relevance of X-ray methods, particularly crystallography. Cryo-EM is now able to probe proteins as small as hemoglobin (64 kDa), while avoiding the crystallization bottleneck entirely. 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.
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