Single-particle electron cryomicroscopy (cryo-EM) has enabled rapid advances in our understanding of membrane protein structure and function. The primary goal during the development of cryo-EM was to perform experiments equivalent to X-ray crystallography, but without needing to crystallize the protein of interest first. However, exciting recent progress in single-particle cryo-EM has come from relaxing assumptions and constraints related to the homogeneity of samples. These assumptions and constraints, which were necessary for crystallization, include that all molecules imaged have the same composition and are in the same conformation, that the specimen consists of only one species, and that the specimen is derived from a solution of isolated protein particles. Here, I discuss the study of membrane protein complexes within lipid bilayers by single-particle cryo-EM. I point out the value and recently achieved capability of studying membrane proteins in lipid vesicles, and in particular endogenous membrane proteins in vesicles prepared from their native lipid bilayer.
