Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding. Author Matthew Doyle, John Jimah, Tyrone Dowdy, Shannon Ohlemacher, Mioara Larion, Jenny Hinshaw, Harris Bernstein Publication Year 2022 Type Journal Article Abstract Transmembrane β barrel proteins are folded into the outer membrane (OM) of Gram-negative bacteria by the β barrel assembly machinery (BAM) via a poorly understood process that occurs without known external energy sources. Here, we used single-particle cryo-EM to visualize the folding dynamics of a model β barrel protein (EspP) by BAM. We found that BAM binds the highly conserved "β signal" motif of EspP to correctly orient β strands in the OM during folding. We also found that the folding of EspP proceeds via "hybrid-barrel" intermediates in which membrane integrated β sheets are attached to the essential BAM subunit, BamA. The structures show an unprecedented deflection of the membrane surrounding the EspP intermediates and suggest that β sheets progressively fold toward BamA to form a β barrel. Along with in vivo experiments that tracked β barrel folding while the OM tension was modified, our results support a model in which BAM harnesses OM elasticity to accelerate β barrel folding. Keywords Escherichia coli, Escherichia coli Proteins, Protein Folding, Bacterial Outer Membrane Proteins, Cryoelectron Microscopy Journal Cell Volume 185 Issue 7 Pages 1143-1156.e13 Date Published 2022 Mar 31 ISSN Number 1097-4172 DOI 10.1016/j.cell.2022.02.016 Alternate Journal Cell PMCID PMC8985213 PMID 35294859 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML