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- EMDB-1763: 3D reconstruction of the type ii secretin GspD from Vibrio cholera -

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Basic information

Entry
Database: EMDB / ID: EMD-1763
Title3D reconstruction of the type ii secretin GspD from Vibrio cholera
Map datacryoEM 3D reconstruction of the type ii secretin GspD from vibrio cholera
Sample
  • Sample: Type ii secretin GspD from Vibrio cholera
  • Protein or peptide: General Secretion Protein D
KeywordsSecretin / GspD / EpsD / type 2 secretion system / T2SS / outermembrane channel
Function / homologyType II secretion system protein GspD, conserved site / protein secretion
Function and homology information
Biological speciesVibrio cholerae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 19.0 Å
AuthorsReichow SL / Korotkov KV / Hol WGJ / Gonen T
CitationJournal: Nat Struct Mol Biol / Year: 2010
Title: Structure of the cholera toxin secretion channel in its closed state.
Authors: Steve L Reichow / Konstantin V Korotkov / Wim G J Hol / Tamir Gonen /
Abstract: The type II secretion system (T2SS) is a macromolecular complex spanning the inner and outer membranes of Gram-negative bacteria. Remarkably, the T2SS secretes folded proteins, including multimeric ...The type II secretion system (T2SS) is a macromolecular complex spanning the inner and outer membranes of Gram-negative bacteria. Remarkably, the T2SS secretes folded proteins, including multimeric assemblies such as cholera toxin and heat-labile enterotoxin from Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. The major outer membrane T2SS protein is the 'secretin' GspD. Cryo-EM reconstruction of the V. cholerae secretin at 19-Å resolution reveals a dodecameric structure reminiscent of a barrel, with a large channel at its center that contains a closed periplasmic gate. The GspD periplasmic domain forms a vestibule with a conserved constriction, and it binds to a pentameric exoprotein and to the trimeric tip of the T2SS pseudopilus. By combining our results with structures of the cholera toxin and T2SS pseudopilus tip, we provide a structural basis for a possible secretion mechanism of the T2SS.
History
DepositionJul 14, 2010-
Header (metadata) releaseJul 23, 2010-
Map releaseNov 12, 2010-
UpdateNov 12, 2010-
Current statusNov 12, 2010Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3.4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1763.gif

EMD-1763.jpg

Downloads & links

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Map

FileDownload / File: emd_1763.map.gz / Format: CCP4 / Size: 12.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationcryoEM 3D reconstruction of the type ii secretin GspD from vibrio cholera
Voxel sizeX=Y=Z: 2.54 Å
Density
Contour LevelBy AUTHOR: 3.4 / Movie #1: 3.4
Minimum - Maximum-2.97366 - 10.769500000000001
Average (Standard dev.)0.00142846 (±0.999409)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 381 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.542.542.54
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z381.000381.000381.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ454586
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS150150150
D min/max/mean-2.97410.7700.001

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Supplemental data

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Sample components

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Entire : Type ii secretin GspD from Vibrio cholera

EntireName: Type ii secretin GspD from Vibrio cholera
Components
  • Sample: Type ii secretin GspD from Vibrio cholera
  • Protein or peptide: General Secretion Protein D

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Supramolecule #1000: Type ii secretin GspD from Vibrio cholera

SupramoleculeName: Type ii secretin GspD from Vibrio cholera / type: sample / ID: 1000 / Oligomeric state: Dodecamer / Number unique components: 1
Molecular weightExperimental: 900 KDa / Theoretical: 900 KDa / Method: Size-exclusion chromatography

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Macromolecule #1: General Secretion Protein D

MacromoleculeName: General Secretion Protein D / type: protein_or_peptide / ID: 1 / Name.synonym: GspD / Number of copies: 12 / Oligomeric state: Dodecamer / Recombinant expression: Yes
Source (natural)Organism: Vibrio cholerae (bacteria) / Location in cell: Membrane
Molecular weightExperimental: 900 KDa / Theoretical: 900 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pMMB710
SequenceGO: protein secretion
InterPro: Type II secretion system protein GspD, conserved site

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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

Concentration0.05 mg/mL
BufferpH: 7.8
Details: 20mM Tris-HCl pH 7.8, 300mM NaCl, N-dodecyl-N,N-dimethylammonio-1-propanesulfonate (SB3-12)
GridDetails: Holey carbon grid with cont. carbon overlayed
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: Blot with filter paper and plunge to liquid ethane

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 51159 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Gatan / Specimen holder model: GATAN LIQUID NITROGEN
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.9 µm
Details: Images were binned 2 times after scanning to yield a final pixel size of 2.54 angstrom
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTFTILT
Final reconstructionApplied symmetry - Point group: C12 (12 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: FREALIGN / Number images used: 10000
DetailsParticles were selected by hand

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