11The ESX (or Type VII) secretion systems are protein export systems in mycobacteria and 12 many Gram-positive bacteria that mediate a broad range of functions including virulence, 13 conjugation, and metabolic regulation. These systems translocate folded dimers of WXG100-14 superfamily protein substrates across the cytoplasmic membrane; however, the architecture and 15 mechanism of translocation has remained elusive. We report the cryo-electron microscopy 16 structure of an ESX-3 system, purified using an epitope tag inserted with recombineering into the 17 model organism Mycobacterium smegmatis. The structure reveals two large a-helical membrane 18 pores of sufficient diameter to secrete folded substrates. A complex, asymmetric, multimeric 19 cytoplasmic domain is poised to gate and regulate the pore's function. Our study provides 20 mechanistic insights into the ESX systems and will guide structure-based design of drugs 21 targeting this unique bacterial translocon. 22 23 One Sentence Summary: The structure of the ESX-3 secretion system reveals a pore of 24 sufficient size for the transit of folded substrates and a complex, cytoplasmic regulatory 25 apparatus. 26 65 expression of ESX-3 ( Fig. S2A). Four components of the complex EccB3, EccC3, EccD3 and 66 EccE3 were affinity-purified as a large molecular weight species (Fig. 1B and 1C, Fig. S2B) 67 using a GFP-nanobody and the GFP tag was proteolytically cleaved. 68 The ESX-3 translocon complex was imaged by cryo-EM and reconstructed revealing a 69 dimeric structure which can be divided into four functionally important areas, the transmembrane 70 translocon pore, the cytoplasmic translocon gate, the cytoplasmic motor domain, and the 71