Type IV secretion systems are secretion nanomachines spanning the two membranes of Gram-negative bacteria. Three proteins, VirB7, VirB9, and VirB10 assemble into a 1.05 MDa core spanning the inner and outer membranes. This core consists of 14 copies of each of the proteins and forms two layers, the I and O layers, inserting in the inner and outer membrane, respectively. Here we present the crystal structure of a ~0.6 MDa outer membrane complex containing the entire O-layer. This structure is the largest determined for an outer membrane channel and is also unprecedented in being composed of three proteins. Unexpectedly, this structure identifies VirB10 as the outer membrane channel with a unique hydrophobic double helical trans-membrane region. This structure establishes VirB10 as the only known protein crossing both membranes of Gram-negative bacteria. Comparison of the cryo-EM and crystallographic structures point to conformational changes regulating channel opening and closing.

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