Fig 1.
FlhA from Vibrio parahaemolyticus and SctV from Yersinia enterocolitica assemble into a large complex in the membrane of E. coli.
a. Fluorescence images of E. coli cells expressing GFP tagged Vp-FlhA or Ye-SctV in the cell envelope. Scale bar 5 μm. b. SDS-PAGE of purified Vp-FlhA and Ye-SctV following cleavage of GFP. c. Gel filtration traces of purified Vp-FlhA and Ye-SctV. The grey shaded area indicates fractions collected to make cryoEM grids.
Fig 2.
Cryo-EM volumes of the cytoplasmic domain of SctV and FlhA.
a. 3D reconstruction of Ye-SctV at a resolution of 3.7 Å using D9 symmetry. b. 3D reconstruction of Vp-FlhA at a resolution of 3.8 Å using C9 symmetry. c. Model of the Ye-SctV linker region in the cryo-EM volume (orange) in the context of the adjacent protomer (grey). d. Model of the Vp-FlhA linker region in the cryo-EM volume (blue) in the context of the adjacent protomer (grey).
Table 1.
CryoEM data collection, processing and model statistics.
Fig 3.
The cryo-EM structures of Ye-SctV and Vp-FlhA are in the open state.
a. Overlay of the nonameric rings of the cytoplasmic domains of Ye-SctV and Vp-FlhA with the crystal structure of the Sf-SctV nonameric ring (PDB: 4A5P). b. Overlay of a single subunit of Ye-SctV and Vp-FlhA with St-FlhA in the open state (grey, PDB: 3A5I) and Sf-SctV in the closed state (red, PDB: 4A5P). c. Overlay of Vp-FlhA (light blue) on the SD3 domain of Sf-SctV (red) and overlay (dark blue) on the SD1 and SD2 domains of Sf-SctV illustrating the movement of the linker (arrow) as the protein changes from the open to the closed state.
Fig 4.
Position of the membrane domain of FlhA in the T3SS nanomachine.
a. The volume of Vp-FlhA is shown in red overlaid onto the tomographic reconstruction of the injectisome (EMD-8544). b. Selected 2D class averages of Vp-FlhA show the high level of detail in the cytoplasmic domain and the lower information content in the presumed transmembrane, micelle-embedded, portion. Scale bar is 100A.