High-resolution cryo-EM: the nuts and bolts

•Near-atomic resolution cryo-EM structures of challenging targets can now be obtained.•Advances in sample preparation increases chance of high-resolution structure determination.•Technical advancements in ultra-stable supports have improved specimen stability.•Direct electron detector developments have contributed to improved resolution. Cryogenic electron microscopy (cryo-EM) and single-particle analysis now enables the determination of high-resolution structures of macromolecular assemblies that have resisted X-ray crystallography and other approaches. Successful high-resolution structure determination by cryo-EM always depends on the quality of the protein sample. While structural heterogeneity remains a key challenge for cryo-EM, it also represents a rare opportunity to study the intrinsic conformational flexibility of macromolecular assemblies. Here, we review the key technological advancements that have made this ‘resolution revolution’ possible and give a concise overview of the technical challenges that needed to be overcome to allow high-resolution structure determination.