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- PDB-8ftf: CryoEM strucutre of 33-mer RBM3 of the Salmonella MS-ring -

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Basic information

Entry
Database: PDB / ID: 8ftf
TitleCryoEM strucutre of 33-mer RBM3 of the Salmonella MS-ring
ComponentsFlagellar M-ring protein
KeywordsMOTOR PROTEIN / flagellar / 33-fold / ring-building motif / MS-ring / FliF
Function / homology
Function and homology information


bacterial-type flagellum basal body, MS ring / cytoskeletal motor activity / bacterial-type flagellum-dependent cell motility / plasma membrane
Similarity search - Function
Flagellar M-ring protein FliF / Flagellar M-ring C-terminal / Flagellar M-ring protein C-terminal / Lipoprotein YscJ/Flagellar M-ring protein / Secretory protein of YscJ/FliF family / Flagellar M-ring , N-terminal / AMP-binding enzyme, C-terminal domain superfamily
Similarity search - Domain/homology
Flagellar M-ring protein
Similarity search - Component
Biological speciesSalmonella enterica subsp. enterica serovar Typhimurium (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsSingh, P.S. / Nakagawa, T. / Cecchini, G. / Iverson, T.M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/John E. Fogarty International Center (NIH/FIC)GM61606 United States
CitationJournal: 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.
History
DepositionJan 12, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 31, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Flagellar M-ring protein
B: Flagellar M-ring protein
C: Flagellar M-ring protein
D: Flagellar M-ring protein
E: Flagellar M-ring protein
F: Flagellar M-ring protein
G: Flagellar M-ring protein
H: Flagellar M-ring protein
I: Flagellar M-ring protein
J: Flagellar M-ring protein
K: Flagellar M-ring protein
L: Flagellar M-ring protein
M: Flagellar M-ring protein
N: Flagellar M-ring protein
O: Flagellar M-ring protein
P: Flagellar M-ring protein
Q: Flagellar M-ring protein
R: Flagellar M-ring protein
S: Flagellar M-ring protein
T: Flagellar M-ring protein
V: Flagellar M-ring protein
W: Flagellar M-ring protein
X: Flagellar M-ring protein
Y: Flagellar M-ring protein
Z: Flagellar M-ring protein
AA: Flagellar M-ring protein
BA: Flagellar M-ring protein
CA: Flagellar M-ring protein
DA: Flagellar M-ring protein
EA: Flagellar M-ring protein
FA: Flagellar M-ring protein
GA: Flagellar M-ring protein
HA: Flagellar M-ring protein


Theoretical massNumber of molelcules
Total (without water)2,022,75633
Polymers2,022,75633
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ...
Flagellar M-ring protein


Mass: 61295.645 Da / Num. of mol.: 33
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Gene: fliF, fla AII.1, fla BI, STM1969 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P15928

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: MS-ring of the flagellar complex / Type: COMPLEX
Details: CryoEM strucutre of the 33-mer RBM3 region of the Salmonella MS-ring
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 2.5 MDa / Experimental value: NO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Details: 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1 % Ana-grade LDAO
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris-HCl1
2100 mMSodium ChlorideNaCl1
30.1 Percentn-Dodecyl-N,N-Dimethylamine-N-Oxide (LDAO)1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Quantifoil grid coated with graphene oxide substrate (Electron Microscopy Sciences) was glow discharged for 5 sec. Purified MS-rings were added to each grid at 4C at 100% humidity. After 30 ...Details: Quantifoil grid coated with graphene oxide substrate (Electron Microscopy Sciences) was glow discharged for 5 sec. Purified MS-rings were added to each grid at 4C 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).
Grid material: GRAPHENE OXIDE / Grid mesh size: 400 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
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 imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC2.15particle selection
4cryoSPARC2.15CTF correction
7UCSF ChimeraXmodel fitting
9PHENIX1.20.1model refinement
10Coot0.9.8.4model refinement
11cryoSPARC2.15initial Euler assignment
12cryoSPARC2.15final Euler assignment
14cryoSPARC4.0.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C33 (33 fold cyclic)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 32042 / Symmetry type: POINT

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