Biological macromolecules and assemblies precisely rearrange their atomic 3D structures to execute cellular functions. Understanding the mechanisms by which these molecular machines operate requires insight into the ensemble of structural states they occupy during the functional cycle. Single-particle cryo-electron microscopy (cryo-EM) has become the preferred method to provide near-atomic resolution, structural information about dynamic biological macromolecules elusive to other structure determination methods. Recent advances in cryo-EM methodology have allowed structural biologists not only to probe the structural intermediates of biochemical reactions, but also to resolve different compositional and conformational states present within the same dataset. This article reviews newly developed sample preparation and single-particle analysis (SPA) techniques for high-resolution structure determination of intrinsically dynamic and heterogeneous samples, shedding light upon the intricate mechanisms employed by molecular machines and helping to guide drug discovery efforts.
Keywords: SPA; cryo-EM; deep learning algorithms; heterogeneity; single-particle analysis; single-particle cryo-electron microscopy.