For decades, high-resolution structural studies of biological macromolecules with masses of <200kDa by cryo-EM single-particle analysis were considered infeasible. It was not until several years after the advent of direct detectors that the overlooked potential of cryo-EM for studying small complexes was first realized. Subsequent advances in sample preparation, imaging, and data processing algorithms have improved our ability to visualize small biological targets. In the past two years alone, nearly two hundred high-resolution structures have been determined of small (<200kDa) macromolecules, the smallest being approximately 39kDa in molecular weight. Here we summarize some salient lessons and strategies for cryo-EM studies of small biological complexes, and also consider future prospects for routine structure determination.
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