A large inner membrane pore defines the ESX translocon

The ESX (or Type VII) secretion systems are protein export systems in mycobacteria and many Gram-positive bacteria that mediate a broad range of functions including virulence, conjugation, and metabolic regulation. These systems translocate folded dimers of WXG100-superfamily protein substrates across the cytoplasmic membrane; however, the architecture and mechanism of translocation has remained elusive. We report the cryo-electron microscopy structure of an ESX-3 system, purified using an epitope tag inserted with recombineering into the model organism . The structure reveals two large -helical membrane pores of sufficient diameter to secrete folded substrates. A complex, asymmetric, multimeric cytoplasmic domain is poised to gate and regulate the pore’s function. Our study provides mechanistic insights into the ESX systems and will guide structure-based design of drugs targeting this unique bacterial translocon.