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- EMDB-29425: CryoEM map of 33-mer RBM3 of the Salmonella MS-ring -

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Open data


ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-29425
TitleCryoEM map of 33-mer RBM3 of the Salmonella MS-ring
Map dataC33 RBM3 Map
Sample
  • Complex: MS-ring of the flagellar complex
    • Protein or peptide: Flagellar M-ring protein
Keywordsflagellar / 33-fold / ring-building motif / MS-ring / FliF / MOTOR PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 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-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29425.png

Downloads & links

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Map

FileDownload / File: emd_29425.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationC33 RBM3 Map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.91 Å/pix.
x 600 pix.
= 544.8 Å		0.91 Å/pix.
x 600 pix.
= 544.8 Å		0.91 Å/pix.
x 600 pix.
= 544.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.908 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.163
Minimum - Maximum-0.46604913 - 1.0953838
Average (Standard dev.)-0.00012513858 (±0.02363827)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 544.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_29425_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: C33 RBM3 Half Map A

Fileemd_29425_half_map_1.map
AnnotationC33 RBM3 Half Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: C33 RBM3 Half Map B

Fileemd_29425_half_map_2.map
AnnotationC33 RBM3 Half Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

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
    • Protein or peptide: Flagellar M-ring protein

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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 / Macromolecule list: all
Details: CryoEM strucutre of the 33-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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Macromolecule #1: Flagellar M-ring protein

MacromoleculeName: Flagellar M-ring protein / type: protein_or_peptide / ID: 1 / Number of copies: 33 / Enantiomer: LEVO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Molecular weightTheoretical: 61.295645 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSATASTATQ PKPLEWLNRL RANPRIPLIV AGSAAVAIVV AMVLWAKTPD YRTLFSNLSD QDGGAIVAQL TQMNIPYRFA NGSGAIEVP ADKVHELRLR LAQQGLPKGG AVGFELLDQE KFGISQFSEQ VNYQRALEGE LARTIETLGP VKSARVHLAM P KPSLFVRE ...String:
MSATASTATQ PKPLEWLNRL RANPRIPLIV AGSAAVAIVV AMVLWAKTPD YRTLFSNLSD QDGGAIVAQL TQMNIPYRFA NGSGAIEVP ADKVHELRLR LAQQGLPKGG AVGFELLDQE KFGISQFSEQ VNYQRALEGE LARTIETLGP VKSARVHLAM P KPSLFVRE QKSPSASVTV TLEPGRALDE GQISAVVHLV SSAVAGLPPG NVTLVDQSGH LLTQSNTSGR DLNDAQLKFA ND VESRIQR RIEAILSPIV GNGNVHAQVT AQLDFANKEQ TEEHYSPNGD ASKATLRSRQ LNISEQVGAG YPGGVPGALS NQP APPNEA PIATPPTNQQ NAQNTPQTST STNSNSAGPR STQRNETSNY EVDRTIRHTK MNVGDIERLS VAVVVNYKTL ADGK PLPLT ADQMKQIEDL TREAMGFSDK RGDTLNVVNS PFSAVDNTGG ELPFWQQQSF IDQLLAAGRW LLVLVVAWIL WRKAV RPQL TRRVEEAKAA QEQAQVRQET EEAVEVRLSK DEQLQQRRAN QRLGAEVMSQ RIREMSDNDP RVVALVIRQW MSNDHE

UniProtKB: Flagellar M-ring protein

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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 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 5 sec.
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 ...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).
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 reconstructionApplied symmetry - Point group: C33 (33 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0.1) / Number images used: 32042
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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