The T4S system outer membrane complex. Ribbon diagram (a) and space-filling cut-away (b) of the tetradecameric complex. TraF_CT, TraO_CT and TraN subunits are colour-coded green, cyan, and magenta, respectively. In b, dimensions and labelling of the various parts of the complex are provided.

Inter-heterotrimeric interactions. a. Schematic diagram of the tetradecamer with emphasis on interactions with the TraF_CT subunit in green (F1). TraF_CT and TraO_CT subunits are shown in circles and hexagons, respectively. Not shown for clarity: TraN subunits (except N14 that makes interactions with F1) and the lever arms of subunits not interacting with F1. b. Ribbon diagram of the F1-interacting TraF_CT subunits (i.e F4, F3, F2, F14, F13, and F12) viewed from the periplasm i.e. turned 180° compared to the view in panel a. The rectangle locates the lever arm of subunit F1. c. Interactions of subunits F2, F3, and F4 with the lever arm of subunit F1. The secondary structures of the lever arm of F1 are labelled. The F2, F3, and F4 subunits are in ribbon representation and labelled accordingly. d. Cut-away side view of the outer membrane complex with the proteins in ribbon diagram representation color-coded as in Fig. 1 but for the N-terminal arms of the TraF_CT subunits in red. e. Schematic diagram of the tetradecamer with emphasis on interactions with the TraO_CT subunit in cyan (O1). Not shown for clarity: TraN subunits (except N1 that makes interactions with O1) and the lever arms of subunits not interacting with O1. f. Ribbon diagram of the subunits shown in colour at left. This view is from the extracellular milieu.

Ribbon diagram of the heterotrimer unit. Structures mentioned in the main text are indicated. The insert locates the shown heterotrimer within the tetradecameric structure. A stereo version of this figure with full secondary structure labelling is provided in Supplementary Fig. 2d.

Trans-membrane region of the T4S system outer membrane complex and proposed mechanism of pore opening/closure. a. Left panel: surface diagram of complex viewed from the extracellular milieu. The two lines define the hydrophobic central region around the pore. Top right panel: same as at left but showing (in yellow) the ring of Trp residues at the base of α2. Bottom right panel: ribbons diagram of the trans-membrane helices with the internal and external ring of helices colour-coded in green and red, respectively. b. Extracellular detection of the FLAG-tag located between helices α2 and α3 of TraF. A FLAG-tag was introduced in the loop between the α2 and α3 helices of TraF as described in methods. Upper and lower left panels: control sample expressing the wild-type T4SS core complex. Upper and lower right panels: cells expressing the FLAG-tagged T4SS core complex. Differential interference contrast images are shown in the upper panels and the corresponding fluorescence images in the lower panels. c. Proposed mechanism for pore opening and closure. The cryo-EM core complex structure (pale yellow) is superimposed on the crystal structure of the outer membrane complex (pale cyan). The N-terminal lever arm shelf is highlighted in orange and red in the EM and crystal structures, respectively. The position of the lipid on TraN/VirB7 is indicated in black dots. TraN/VirB7 is shown in magenta. Proposed conformational changes are illustrated by arrows. The N-terminal sequences linking the lever arms to sequences in the I-layer are shown in dashed lines colour-coded red and orange for the conformations found in the crystal and EM structures, respectively.