Figure 5. Changes between the open and closed state of the T4P machinery and its distribution in situ.

(A and B) Comparisons between the PilQ components of the T4P machinery reveal large conformational changes whereby both gates open and domains N0–N3 (now shown in blue for both states) shift by ∼30 Å towards the cytoplasm on pilus extrusion. Green arrowheads indicate additional protein densities (C1 = proximal to the cytoplasmic membrane, P1 = central periplasmic ring 1, P2 = central periplasmic ring 2). In (B) the structure of the T4P has been docked into the open state. OM, outer membrane; PG, peptidoglycan; CM, cytoplasmic membrane. (C and D) Docking subtomogram averages (purple) into the tomographic volume of a cell reveals the distribution of the closed T4P machinery in situ with respect to the outer membrane (pale yellow) and cytoplasmic membrane (blue). See Video 2 for details. (E) Averaged histogram of nearest-neighbour distance between protein complexes, calculated from 9 cells, with a total of 332 data points. Error bars indicate the standard deviation of the frequency distribution for each minimal distance.

DOI: http://dx.doi.org/10.7554/eLife.07380.008

Figure 5—figure supplement 1. Subtomogram averages from cells with high periplasmic protein content.

Subtomogram averages were calculated for the open and closed state of the T4P machinery from the tomogram shown in Figure 2A–C, where the periplasm contains high-protein levels. The resolution of the subtomogram averages is limited due to the low number of protein complexes available from a single tomogram. Therefore, data for PilQ and protein P2 (green arrowhead) only are shown, which indicate essentially the same large conformational change in domains N0–N3 (blue) as seen in Figure 5A,B.

DOI: http://dx.doi.org/10.7554/eLife.07380.009