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- EMDB-29429: CryoEM map of 34-mer RBM3 of the Salmonella MS-ring -

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Basic information

Entry
Database: EMDB / ID: EMD-29429
TitleCryoEM map of 34-mer RBM3 of the Salmonella MS-ring
Map data
Sample
  • Complex: MS-ring of the flagellar complex
Keywordsflagellar / 34-fold / ring-building motif / MS-ring / FliF / MOTOR PROTEIN
Biological speciesSalmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsSingh PS / Nakagawa T / Cecchini G / Iverson TM
Funding support United States, 1 items
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, 2023-
Header (metadata) releaseMay 31, 2023-
Map releaseMay 31, 2023-
UpdateMay 31, 2023-
Current statusMay 31, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29429.png

Downloads & links

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Map

FileDownload / File: emd_29429.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.36 Å/pix.
x 400 pix.
= 544. Å		1.36 Å/pix.
x 400 pix.
= 544. Å		1.36 Å/pix.
x 400 pix.
= 544. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.36 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.222
Minimum - Maximum-0.6744834 - 1.6774323
Average (Standard dev.)-0.00022677548 (±0.04101838)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 544.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_29429_msk_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_29429_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_29429_half_map_2.map
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : MS-ring of the flagellar complex

EntireName: MS-ring of the flagellar complex
Components
  • Complex: MS-ring of the flagellar complex

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Supramolecule #1: MS-ring of the flagellar complex

SupramoleculeName: MS-ring of the flagellar complex / type: complex / ID: 1 / Parent: 0
Details: CryoEM strucutre of the 32-mer RBM3 region of the Salmonella MS-ring
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Molecular weightTheoretical: 2.5 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Component:
ConcentrationNameFormula
50.0 mMTris-HCl
100.0 mMSodium ChlorideNaCl
0.1 Percentn-Dodecyl-N,N-Dimethylamine-N-Oxide (LDAO)

Details: 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1 % Ana-grade LDAO
GridModel: UltrAuFoil R1.2/1.3 / Material: GRAPHENE OXIDE / Mesh: 400 / Support film - Material: GRAPHENE OXIDE
VitrificationCryogen 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

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

10143

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0.1) / Number images used: 26843
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15)
FSC plot (resolution estimation)

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