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- EMDB-1617: C12 symmetrised 3D reconstruction of the Shigella flexneri T3SS n... -

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Basic information

Entry
Database: EMDB / ID: 1617
TitleC12 symmetrised 3D reconstruction of the Shigella flexneri T3SS needle complex from negatively stained sample electron micrographs
KeywordsShigella flexneri Type III secretion system Needle complex Microbial pathogenesis
SampleShigella flexneri T3SS needle complex
SourceShigella flexneri / bacteria / Dysentery bacillus / シゲラ・フレクスネリ, フレクスナー赤痢菌
Map dataThis is a 3D reconstruction of the Shigella flexneri 'needle complex' from negative stain images, done with C12 symmetry imposed. The resolution is 21-25A.
Methodsingle particle reconstruction, at 25 Å resolution
AuthorsHodgkinson JL / Horsley A / Stabat D / Simon M / Johnson S / da Fonseca PCA / Morris EP / Wall JS / Lea SM / Blocker AJ
CitationNat. Struct. Mol. Biol., 2009, 16, 477-485

Nat. Struct. Mol. Biol., 2009, 16, 477-485 StrPapers
Three-dimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout.
Julie L Hodgkinson / Ashley Horsley / David Stabat / Martha Simon / Steven Johnson / Paula C A da Fonseca / Edward P Morris / Joseph S Wall / Susan M Lea / Ariel J Blocker

DateDeposition: May 1, 2009 / Header (metadata) release: May 20, 2009 / Map release: May 27, 2009 / Last update: Apr 23, 2010

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.2
  • Imaged by UCSF CHIMERA
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.2
  • Imaged by UCSF CHIMERA
  • Download
3D viewer


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Supplemental images

Downloads & links

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Map

Fileemd_1617.map.gz (map file in CCP4 format, 23551 KB)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
182 pix
2.62 Å/pix.
= 476.84 Å
182 pix
2.62 Å/pix.
= 476.84 Å
182 pix
2.62 Å/pix.
= 476.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Voxel sizeX=Y=Z: 2.62 Å
Density
Contour Level:1 (by author), 1.2 (movie #1):
Minimum - Maximum-13.1014 - 9.57075
Average (Standard dev.)-1.06342e-09 (1)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions182182182
Origin-91-91-91
Limit909090
Spacing182182182
CellA=B=C: 476.84 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.622.622.62
M x/y/z182182182
origin x/y/z0.0000.0000.000
length x/y/z476.840476.840476.840
α/β/γ90.00090.00090.000
start NX/NY/NZ-17-17-200
NX/NY/NZ123123401
MAP C/R/S123
start NC/NR/NS-91-91-91
NC/NR/NS182182182
D min/max/mean-13.1019.571-0.000

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Supplemental data

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Sample components

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Entire Shigella flexneri T3SS needle complex

EntireName: Shigella flexneri T3SS needle complex
Details: Affinity purified using His6 tag on MxiG N-term Contains detergent (see Zenk et al., 2007)
Number of components: 8
Oligomeric State: Large macromolecular complex (total list of components not yet known)
MassExperimental: 3.6 MDa / Measured by: STEM analysis

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Component #1: protein, Spa24

ProteinName: Spa24 / a.k.a: Spa24 / Oligomeric Details: Unknown
Details: This is the MW of the monomer, which must be cleaved in its cytoplasmic portion during T3SS biogenesis.
Recombinant expression: No
MassTheoretical: 240 kDa
SourceSpecies: Shigella flexneri / bacteria / Dysentery bacillus / シゲラ・フレクスネリ, フレクスナー赤痢菌
Strain: Shigella flexneri M90T
Source (natural)Organelle: Type III secretion system / Location in cell: inner membrane protein / Cell: Gram-negative bacterium
External referencesInterPro: InterPro: 005773 / Gene Ontology: GO: 0009306

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Component #2: protein, Spa40

ProteinName: Spa40 / a.k.a: Spa40 / Oligomeric Details: Unknown
Details: This is the MW of the monomer, which must be cleaved in its cytoplasmic portion during T3SS biogenesis.
Recombinant expression: No
MassTheoretical: 400 kDa
SourceSpecies: Shigella flexneri / bacteria / Dysentery bacillus / シゲラ・フレクスネリ, フレクスナー赤痢菌
Strain: Shigella flexneri M90T
Source (natural)Organelle: Type III secretion system / Location in cell: polytopic-inner membrane protein / Cell: Gram-negative bacterium
External referencesGene Ontology: GO: 0009306 / InterPro: InterPro: 006307

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Component #3: protein, MxiM

ProteinName: MxiM / a.k.a: MxiM / Details: This is the MW of the monomer. / Recombinant expression: No / Number of Copies: 12
MassTheoretical: 150 kDa
SourceSpecies: Shigella flexneri / bacteria / Dysentery bacillus / シゲラ・フレクスネリ, フレクスナー赤痢菌
Strain: Shigella flexneri M90T
Source (natural)Organelle: Type III secretion system / Location in cell: outside face of outer membrane / Cell: Gram-negative bacterium
External referencesGene Ontology: GO: 0009279

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Component #4: protein, MxiI

ProteinName: MxiI / a.k.a: MxiI / Oligomeric Details: Helical polymer / Details: This is the MW of the monomer. / Number of Copies: 10 / Recombinant expression: No
MassTheoretical: 100 kDa
SourceSpecies: Shigella flexneri / bacteria / Dysentery bacillus / シゲラ・フレクスネリ, フレクスナー赤痢菌
Strain: Shigella flexneri M90T
Source (natural)Organelle: Type III secretion system / Location in cell: periplasmic space / Cell: Gram-negative bacterium
External referencesGene Ontology: GO: 0015031

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Component #5: protein, MxiG

ProteinName: MxiG / a.k.a: MxiG / Oligomeric Details: Probable homo-24mer / Details: This is the MW of the monomer. / Number of Copies: 24 / Recombinant expression: No
MassTheoretical: 430 kDa
SourceSpecies: Shigella flexneri / bacteria / Dysentery bacillus / シゲラ・フレクスネリ, フレクスナー赤痢菌
Strain: Shigella flexneri M90T
Source (natural)Organelle: Type III secretion system / Location in cell: spans inner membrane / Cell: Gram-negative bacterium
External referencesGene Ontology: GO: 0009405

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Component #6: protein, MxiJ

ProteinName: MxiJ / a.k.a: MxiJ / Oligomeric Details: Probable homo-dodecamer
Details: This is the MW of the monomer with its N-terminal lipid modification site processed (but MW of lipid moiety unknown).
Recombinant expression: No / Number of Copies: 12
MassTheoretical: 250 kDa
SourceSpecies: Shigella flexneri / bacteria / Dysentery bacillus / シゲラ・フレクスネリ, フレクスナー赤痢菌
Strain: Shigella flexneri M90T
Source (natural)Organelle: Type III secretion system / Location in cell: mostly periplasmic face of inner membrane / Cell: Gram-negative bacterium
External referencesGene Ontology: GO: 0009306 / InterPro: InterPro: 006182

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Component #7: protein, MxiH

ProteinName: MxiH / a.k.a: MxiH / Oligomeric Details: helical polymer / Details: This is the MW of the monomer. / Recombinant expression: No / Number of Copies: 120
MassTheoretical: 90 kDa
SourceSpecies: Shigella flexneri / bacteria / Dysentery bacillus / シゲラ・フレクスネリ, フレクスナー赤痢菌
Strain: Shigella flexneri M90T
Source (natural)Organelle: Type III secretion system / Location in cell: Extracellular / Cell: Gram-negative bacterium
External referencesInterPro: InterPro: 011841 / Gene Ontology: GO: 0009405

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Component #8: protein, MxiD

ProteinName: MxiD / a.k.a: MxiD / Oligomeric Details: homo-dodecamer
Details: This is the MW of the monomer without it's signal sequence included
Recombinant expression: No / Number of Copies: 12
MassTheoretical: 620 kDa
SourceSpecies: Shigella flexneri / bacteria / Dysentery bacillus / シゲラ・フレクスネリ, フレクスナー赤痢菌
Strain: Shigella flexneri M90T
Source (natural)Organelle: Type III secretion system / Location in cell: Outer membrane / Cell: Gram-negative bacterium
External referencesInterPro: InterPro: 003522 / Gene Ontology: GO: 0009306

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Experimental details

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Sample preparation

Specimen stateparticle
Sample solutionSpecimen conc.: 0.05 mg/ml
Buffer solution: 10mM Tris pH 8, 0.1% Triton X-100 and 1mM EDTA buffer
pH: 8
Support film400 mesh copper grids (Athene) covered with holey carbon film over which thin plain carbon was laid were used
Stainingnegatively stained in unbuffered 2% aqueous uranyl acetate
VitrificationInstrument: NONE / Cryogen name: NONE

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Electron microscopy imaging

ImagingMicroscope: FEI/PHILIPS CM200FEG / Details: Low-dose mode was used
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 20 e/Å2 / Illumination mode: SPOT SCAN
LensMagnification: 50000 X (nominal), 48600 X (calibrated)
Astigmatism: objective lens astigmatism was corrected at 100,000 times magnification
Cs: 2.2 mm / Imaging mode: BRIGHT FIELD / Defocus: 700 - 900 nm
Specimen HolderHolder: eucentric / Model: OTHER
CameraDetector: KODAK SO-163 FILM

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Image acquisition

Image acquisitionNumber of digital images: 126 / Scanner: NIKON SUPER COOLSCAN 9000 / Sampling size: 6.35 microns / Bit depth: 16 / OD range: 1

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Image processing

ProcessingMethod: single particle reconstruction / Number of class averages: 4 / Number of projections: 41
Details: Numbers of particles given are for final reconstruction. We started with over 3000. Particles were selected using Ximdisp software, cut out to an initial box size of 400 by 400 pixel and coarsened to 2.62A per pixel using Label prior to further processing.
Applied symmetry: C12 (12 fold cyclic)
3D reconstructionAlgorithm: weighted back projection / Euler angles: Imagic beta 87-93 degrees gamma 0-30 degrees / Software: SPIDER IMAGIC5
Details: Maps were generated from 41 individual images (selected through image processing out of an initial pool of 3000)
Resolution: 25 Å / Resolution method: FSC at 3-sigma and 0.5 cut-offs

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