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- EMDB-10653: Structure of the core of the flagellar export apparatus from Vibr... -

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Basic information

Entry
Database: EMDB / ID: EMD-10653
TitleStructure of the core of the flagellar export apparatus from Vibrio mimicus, the FliPQR-FlhB, in amphipol A8-35
Map data
Sample
  • Complex: Complex of Vibrio mimicus FliP, FliQ, FliR, FlhB.
Biological speciesVibrio mimicus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsKuhlen L / Johnson S / Deme JC / Lea SM
Funding support United Kingdom, 5 items
OrganizationGrant numberCountry
Wellcome Trust201536 United Kingdom
Wellcome Trust100298 United Kingdom
Wolfson FoundationWL160052 United Kingdom
Wellcome Trust109136 United Kingdom
Medical Research Council (MRC, United Kingdom)M011984 United Kingdom
CitationJournal: Nat Commun / Year: 2020
Title: The substrate specificity switch FlhB assembles onto the export gate to regulate type three secretion.
Authors: Lucas Kuhlen / Steven Johnson / Andreas Zeitler / Sandra Bäurle / Justin C Deme / Joseph J E Caesar / Rebecca Debo / Joseph Fisher / Samuel Wagner / Susan M Lea /
Abstract: Protein secretion through type-three secretion systems (T3SS) is critical for motility and virulence of many bacteria. Proteins are transported through an export gate containing three proteins ...Protein secretion through type-three secretion systems (T3SS) is critical for motility and virulence of many bacteria. Proteins are transported through an export gate containing three proteins (FliPQR in flagella, SctRST in virulence systems). A fourth essential T3SS protein (FlhB/SctU) functions to "switch" secretion substrate specificity once the growing hook/needle reach their determined length. Here, we present the cryo-electron microscopy structure of an export gate containing the switch protein from a Vibrio flagellar system at 3.2 Å resolution. The structure reveals that FlhB/SctU extends the helical export gate with its four predicted transmembrane helices wrapped around FliPQR/SctRST. The unusual topology of the FlhB/SctU helices creates a loop wrapped around the bottom of the closed export gate. Structure-informed mutagenesis suggests that this loop is critical in gating secretion and we propose that a series of conformational changes in the T3SS trigger opening of the gate through interactions between FlhB/SctU and FliPQR/SctRST.
History
DepositionJan 31, 2020-
Header (metadata) releaseMar 25, 2020-
Map releaseMar 25, 2020-
UpdateMar 25, 2020-
Current statusMar 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.007
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.007
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10653.png

Downloads & links

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Map

FileDownload / File: emd_10653.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.822 Å
Density
Contour LevelBy AUTHOR: 0.007 / Movie #1: 0.007
Minimum - Maximum-0.0097648725 - 0.02780139
Average (Standard dev.)0.0000062217 (±0.0017154317)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 210.432 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8220.8220.822
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z210.432210.432210.432
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0100.0280.000

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Supplemental data

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Half map: Half map 2

Fileemd_10653_half_map_1.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_10653_half_map_2.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of Vibrio mimicus FliP, FliQ, FliR, FlhB.

EntireName: Complex of Vibrio mimicus FliP, FliQ, FliR, FlhB.
Components
  • Complex: Complex of Vibrio mimicus FliP, FliQ, FliR, FlhB.

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Supramolecule #1: Complex of Vibrio mimicus FliP, FliQ, FliR, FlhB.

SupramoleculeName: Complex of Vibrio mimicus FliP, FliQ, FliR, FlhB. / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Vibrio mimicus (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightExperimental: 250 KDa

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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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 48.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 137136
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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