Cryo-EM: beyond the microscope.

The pace at which cryo-EM is being adopted as a mainstream tool in structural biology has continued unabated over the past year. Initial successes in obtaining near-atomic resolution structures with cryo-EM were enabled to a large extent by advances in microscope and detector technology. Here, we review some of the complementary technical improvements that are helping sustain the cryo-EM revolution. We highlight advances in image processing that permit high resolution structure determination even in the presence of structural and conformational heterogeneity. We also review selected examples where biochemical strategies for membrane protein stabilization facilitate cryo-EM structure determination, and discuss emerging approaches for further improving the preparation of reliable plunge-frozen specimens.