Extended Data Fig. 8. Details of the OMCC proteins and description of extra-densities that could not be ascribed.

a, Structure of the O-layer and I-layer. All proteins are in ribbon, with TrwH/VirB7, TrwF/VirB9 and TrwE/VirB10 shown in magenta, light blue and green, respectively. Dimensions of interest are reported. Proteins constituting the shown complexes are indicated. Upper left: side view of the O-layer. Upper right: top view of the O-layer. Lower left: side view of the I-layer. Lower right: top view of the I-layer. b, Secondary structure definition of TrwE/VirB10_CTD (left), TrwF/VirB9_CTD (middle) and TrwH/VirB7 (right). The ribbon for each protein is coloured in rainbow colours from dark blue for the N-terminus to red for the C-terminus. All secondary structures are labelled. Note that loop between α3 and α4 of TrwE/VirB10_CTD is disordered and, as a result, these helices appear to insert half-way through the membrane. However, in Chandran et al. (2009)²³, we showed that the loop connecting the two helices is accessible from the surface of the bacterium and therefore completes the TM region and emerges out to the bacterial cell surface. c, Superposition of the structures of the hetero-trimeric unit of the O-layer from pKM101 (grey) and R388 (light blue, green and magenta for TrwE/VirB10_CTD, TrwF/VirB9_CTD, and TrwH/VirB7, respectively). The two heterotrimers superimpose with an RMSD in Cα of 0.8 Å. The various parts of the heterotrimeric complex are shown and labelled. d, Superposition of the structure of the hetero-dimeric unit of the I-layer from R388 (TrwF/VirB9_NTD (light blue) bound to α1 of TrwE/VirB10_NTD (green)) and that of the same region of the Xanthomonas citri I-layer (grey). The two structures superimpose with an RMSD in Cα of 0.9 Å. e, The interfaces between TrwF/VirB9_NTD subunits (upper panel; the domain is in rainbow colour from N- to C-terminus) and between α1 of TrwE/VirB10_NTD and two TrwF/VirB9_NTD (lower panel). Secondary structures contributing residues to the interfaces are labelled. f, Insertion of two additional TrwF/VirB9_NTD-α1TrwE/VirB10_NTD complexes diametrically opposite within the I-layer. Left: the OMCC C1 3.28 Å map shown in grey with TrwF/VirB9 linkers between the O- and I-layers shown in purple (0.17 σ level). Dashed line box indicates the section of density shown at right. Right: top view of the I-layer map section indicated by the dashed line box at left. The two inserted TrwF/VirB9_NTD domains are recognizable because they do not have the linker connecting their NTDs (I-layer) to their CTDs (O-layer). g, Interactions between TrwI/VirB6 (the base of the Stalk) with TrwM/VirB3 and TrwK/VirB4 (in the IMC). TrwM/VirB3 and TrwI/VirB6 are shown in pale green and red ribbon, respectively, while two central TrwK/VirB4 subunits are shown in dark blue and sky blue ribbon and semi-transparent surface. Secondary structures contributing interacting residues are labelled. Inset: overview of the location of the zoomed-up structure shown in main panel. h, Additional densities (shown in semi-transparent grey) observed in the Arches and the IMC potentially corresponding to a fourth molecule of TrwG/VirB8. Additional density in the IMC-Arches-Stalk C1 6.18 Å map (Extended Data Fig. 3f) was observed forming a helix tube bound to the 3-helices bundle of the TrwG/VirB8_tails. Correspondingly, an additional density was observed near the TrwG/VirB8_peri ring. This may indicate the presence of a fourth TrwG/VirB8 subunit (shown in magenta ribbon). Finally, there is density between the TrwG/VirB8_tails and the TrwG/VirB8_peri domains, which we hypothesize might be formed by the residues in between the two domains (residues 62-95; Extended Data Fig. 1b). However, the densities were too poor to be assigned and we remain unsure as to potential interpretations and assignments. i, Extra density also observed in the IMC-Arches-Stalk C1 6.18 Å map (Extended Data Fig. 3f). Left: overall structure of the T4SS with a dashed lined box showing the location of the extra density. Proteins are colour-coded as in Fig. 1d and are in ribbon representation, except for TrwE/VirB10 which is in surface representation. Right: zoom-in on the region of the structure shown in the dashed lined box shown at left. Two extra densities (in green) are seen at σ 0.04 which merge into one at σ 0.02. These indicate a structure that makes contact with TrwG/VirB8_peri and that is flexibly (shown in dashed lines) connected to another structure that makes contact with the TrwI/VirB6 TM helices and with the two subunits of the TrwK/VirB4 dimer. The density was too poor to be assigned but could correspond to TrwE/VirB10_NTD, which is known to not only make a major part of the OMCC but also has an IM TM and a cytoplasmic tail that, in other T4SSs, has been known to interact with VirB8, VirB6 and VirB4^70–72. However, it could be that this stretch of density may correspond to different proteins.