(A and B) Tomographic slices show close-up views of the T4P machinery with assembled pili (white arrowheads). Scale bar = 50 nm. (C) Upper panel, a tomographic slice of the pilus shows that the structure is periodic. Scale bar = 20 nm. A slice through the subtomogram average (inset) shows the repeat more clearly. Scale bar = 5 nm. Lower panels, power spectra of the tomographic slice (left) and the average (right) depict layer lines (orange arrows) at a distance of 1/(49 Å) from the equator (green arrow), corresponding to a helical pitch of 4.9 nm. The contrast has been inverted. (D) Subtomogram average of the ∼3 nm wide T. thermophilus pilus (green, top panel), compared to the previously determined ∼6 nm wide structure from N. gonorrhoeae (blue, bottom panel, EMDB 1236) (Craig et al., 2006). (E) Subtomogram average (left panel) and 3D surface rendering (right panel) of the T4P machinery in the open state with the central pilus (green). The putative N0–N5 (Burkhardt et al., 2012) domains of PilQ are marked. Additional protein densities distinct from PilQ (green arrowheads; C1 = proximal to the cytoplasmic membrane, P1 = central periplasmic ring 1, P2 = central periplasmic ring 2) are also shown. Compared to the closed state of the complex, additional protein densities (left panel, yellow arrowheads) are visible in the cytoplasm. See Video 1 for details. OM, outer membrane; PG, peptidoglycan; CM, cytoplasmic membrane.