In situ and high‐resolution cryo‐EM structure of a bacterial type VI secretion system membrane complex

Bacteria have evolved macromolecular machineries that secrete effectors and toxins to survive and thrive in diverse environments. The type VI secretion system (T6SS) is a contractile machine that is related to Myoviridae phages. It is composed of a phage tail‐like structure inserted in the bacterial cell envelope by a membrane complex (MC) comprising the TssJ, TssL and TssM proteins. We previously reported the low‐resolution negative‐stain electron microscopy structure of the enteroaggregative Escherichia coli MC and proposed a rotational 5‐fold symmetry with a TssJ:TssL:TssM stoichiometry of 2:2:2. Here, cryo‐electron tomography analyses of the T6SS MC confirm the 5‐fold symmetry in situ and identify the regions of the structure that insert into the bacterial membranes. A high‐resolution model obtained by single‐particle cryo‐electron microscopy highlights new features: five additional copies of TssJ, yielding a TssJ:TssL:TssM stoichiometry of 3:2:2, an 11‐residue loop in TssM, protruding inside the lumen of the MC and constituting a functionally important periplasmic gate, and hinge regions. Based on these data, we propose an updated model on MC structure and dynamics during T6SS assembly and function.