A “Build and Retrieve” methodology to simultaneously solve cryo-EM structures of membrane proteins

Single-particle cryo-electron microscopy (cryo-EM) has become a powerful technique in the field of structural biology. However, the inability to reliably produce pure, homogeneous membrane protein samples significantly hampers the progress of their structural determination. Here, we develop a bottom-up iterative method, designated “Build and Retrieve” (BaR), that allows us to identify and solve cryo-EM structures of a variety of inner and outer membrane proteins, including membrane protein complexes of different sizes and dimensions, from a heterogeneous, impure protein sample. We also employ the BaR methodology to elucidate structural information from E. coli K12 crude membrane and raw lysate. Our work demonstrates that it is possible to solve high-resolution structures of a number of relatively small (< 100 kDa) and less abundant (< 10%) unidentified membrane proteins within a single, heterogeneous sample. Importantly, these results highlight the potential of cryo-EM for systems structural proteomics.