Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding

SUMMARY Transmembrane β-barrel proteins are folded into the o uter m embrane (OM) of Gram-negative bacteria by the β- b arrel a ssembly m achine (BAM) via an unexplained 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 remarkable “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 towards 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.