National Institutes of Health/John E. Fogarty International Center (NIH/FIC)
GM61606
United States
Citation
Journal: PLoS One / Year: 2023 Title: CryoEM structure of a post-assembly MS-ring reveals plasticity in stoichiometry and conformation. Authors: Prashant K Singh / Gary Cecchini / Terunaga Nakagawa / T M Iverson / Abstract: The flagellar motor supports bacterial chemotaxis, a process that allows bacteria to move in response to their environment. A central feature of this motor is the MS-ring, which is composed entirely ...The flagellar motor supports bacterial chemotaxis, a process that allows bacteria to move in response to their environment. A central feature of this motor is the MS-ring, which is composed entirely of repeats of the FliF subunit. This MS-ring is critical for the assembly and stability of the flagellar switch and the entire flagellum. Despite multiple independent cryoEM structures of the MS-ring, there remains a debate about the stoichiometry and organization of the ring-building motifs (RBMs). Here, we report the cryoEM structure of a Salmonella MS-ring that was purified from the assembled flagellar switch complex (MSC-ring). We term this the 'post-assembly' state. Using 2D class averages, we show that under these conditions, the post-assembly MS-ring can contain 32, 33, or 34 FliF subunits, with 33 being the most common. RBM3 has a single location with C32, C33, or C34 symmetry. RBM2 is found in two locations with RBM2inner having C21 or C22 symmetry and an RBM2outer-RBM1 having C11 symmetry. Comparison to previously reported structures identifies several differences. Most strikingly, we find that the membrane domain forms 11 regions of discrete density at the base of the structure rather than a contiguous ring, although density could not be unambiguously interpreted. We further find density in some previously unresolved areas, and we assigned amino acids to those regions. Finally, we find differences in interdomain angles in RBM3 that affect the diameter of the ring. Together, these investigations support a model of the flagellum with structural plasticity, which may be important for flagellar assembly and function.
Supramolecule #1: MS-ring of the flagellar complex
Supramolecule
Name: MS-ring of the flagellar complex / type: complex / ID: 1 / Parent: 0 Details: CryoEM strucutre of the 33-mer RBM3 region of the Salmonella MS-ring
Details: 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1 % Ana-grade LDAO
Grid
Model: UltrAuFoil R1.2/1.3 / Material: GRAPHENE OXIDE / Mesh: 400 / Support film - Material: GRAPHENE OXIDE
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV Details: Purified MS-rings were added to each grid at 277.15K at 100% humidity. After 30 sec of incubation, blotting was performed for 12 sec. The grid was then plunged into liquid ethane using a ...Details: Purified MS-rings were added to each grid at 277.15K at 100% humidity. After 30 sec of incubation, blotting was performed for 12 sec. The grid was then plunged into liquid ethane using a Vitrobot Mark IV system (Thermo Fisher).
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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