Cryo-EM studies of membrane proteins at 200 keV

Single-particle cryogenic electron-microscopy (cryo-EM) has emerged as a powerful technique for the structural character-isation of membrane proteins, especially for targets previously thought to be intractable. Taking advantage of the latest hard -and software developments, high-resolution three-dimensional (3D) reconstructions of membrane proteins by cr yo-EM has become routine, with 300-kV transmission electron micro-scopes (TEMs) being the current standard. The use of 200-kV cryo-TEMs is gaining increasingly prominence, showing the capabilities of reaching better than 2 angstrom resolution for soluble proteins and better than 3 angstrom resolution for membrane proteins. Here, we highlight the challenges working with membrane proteins and the impact of cryo-EM, and review the technical and practical benefits, achievements and limitations of imaging at lower electron acceleration voltages.