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. 2022 Jun 22;607(7917):191–196. doi: 10.1038/s41586-022-04859-y

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 6 — a and b, Assessment of map resolution and quality for the TrwK/VirB4_unbound dimer (a) and trimer of dimers (b) structures. The local resolution variations of the corresponding map (left), the overall resolution derived from Fourier Shell Correlation (FSC) (upper right), the angular distribution (middle right) and a representative region of the electron density map with the final model of the TrwK/VirB4_unbound model built in it (lower right) are shown. Local resolution was calculated using CRYOSPARC and coloured as indicated in the scale below the map. The FSC plot shows curves for correlation between 2 independently refined half-maps with no mask (blue), spherical mask (green), loose mask (red), tight mask (cyan) and corrected (purple). Cut-off 0.143 (blue line) was used for average resolution estimation. The final model in representative regions of the maps is shown in magenta stick and ribbon representation for the dimer and trimer of dimers, respectively. c, Secondary structure definition of TrwK/VirB4 (Left), TrwM/VirB3 (Middle) and TrwG/VirB8_tails (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. The IM is shown as a grey rectangle. d, Superposition of TrwK/VirB4_central and TrwK/VirB4_outside subunits of the IMC protomer. The two structures are very similar (RMSD in Cα position of 1.6 Å). Regions of differences between the two structures are indicated. e, Details of the interactions between two adjacent TrwK/VirB4_central subunits within the central hexamer. The two subunits are both shown in ribbon but coloured dark and sky blue, respectively. All secondary structures involved in the interaction are shown. Without the structure of ATP-bound TrwK/VirB4, it is unclear whether the TrwK/VirB4_central hexamer is in an active form or conformational changes are required to transition into one. f, Superposition of the TrwK/VirB4 dimeric unit of the T4SS (TrwK/VirB4_central and TrwK/VirB4_outside in blue and cyan, respectively, as in Fig. 2) onto the dimeric unit of TrwK/VirB4_unbound (in magenta and pink). These two structures superimpose very well with an RMSD in Cα position of 1.2 Å. g, Assembly of TrwK/VirB4_unbound. In TrwK/VirB4_unbound, three dimer units (shown here in magenta and pink, one of which is surrounded by a rectangle) come together in a roughly head to tail manner to form a trimer of dimers. Left: top view of the trimer of dimers structure. Right: schematic diagram showing the trimer of dimers configuration of TrwK/VirB4_unbound. h, Superposition of the TrwK/VirB4_unbound trimer of dimers (in magenta and pink) and the T4SS TrwK/VirB4 hexamer of dimers (in grey except for the TrwK/VirB4 dimer used for superposition which is shown in cyan and blue for TrwK/VirB4_outside and TrwK/VirB4_central, respectively). Left: the two types of assembly are superposed using the superposed dimeric units shown in panel f as a guide. In this superposition, the TrwK/VirB4_unbound trimer of dimers can be observed in an off-centered position relative to the T4SS TrwK/VirB4 hexamer of dimers. Right: two of the TrwK/VirB4_unbound trimers of dimers superposed on two diametrically opposite TrwK/VirB4_central-TrwK/VirB4_outside dimers results in a double-barrelled architecture reminiscent of that observed in the NSEM double-barrelled structure by Low et al. (2014)⁸ where two off-centered barrels (also trimers of dimers) were observed side by side as shown in Extended Data Fig. 1a, left panel. i, Docking of two TrwK/VirB4_unbound trimers of dimers (green and blue ribbons) into the NSEM double-barrelled structure. Left: NSEM map of the T4SS double-barrelled architecture⁸ contoured at σ 10 (EMD-2567). The region corresponding to TrwK/VirB4 is within the middle and lower tiers densities of each barrel (see Low et al. (2014) for details)⁸. The two dashed arrows indicate where the sections shown at right have been taken. The TrwK/VirB4_unbound NTDs fit well into the middle tier density (fitting correlation 0.59; upper right panel) while the CTDs, which are very flexible because of being unconstrained, protrude out of the lower tier density (fitting correlation 0.29; lower right panel).