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

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. [Display omitted] •BAM binds to β signals to orient outer membrane protein β barrels in the membrane•β barrels fold by passing through an extended β sheet stage while bound to BamA•Extreme membrane remodeling is a key feature of transmembrane β barrel assembly•Outer membrane tension accelerates the final stages of bacterial β barrel folding The folding of the β barrel protein EspP by the β barrel assembly machinery in E. coli involves the progressive conversion of a β sheet into a barrel-like structure in a process that is influenced by membrane tension.