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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: EMD-1617
TitleC12 symmetrised 3D reconstruction of the Shigella flexneri T3SS needle complex from negatively stained sample electron micrographs
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.
Sample
  • Sample: Shigella flexneri T3SS needle complex
  • Protein or peptide: Spa24
  • Protein or peptide: Spa40
  • Protein or peptide: MxiM
  • Protein or peptide: MxiI
  • Protein or peptide: MxiG
  • Protein or peptide: MxiJ
  • Protein or peptide: MxiH
  • Protein or peptide: MxiD
KeywordsShigella flexneri Type III secretion system Needle complex Microbial pathogenesis
Function / homologyYop virulence translocation protein R / Type III exporter system, secretion apparatus protein BsaZ / Type III secretion system outer membrane pore YscC/HrcC / Type III secretion system, needle protein / Flagellar M-ring , N-terminal / protein secretion / : / cell outer membrane / protein transport
Function and homology information
Biological speciesShigella flexneri (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsHodgkinson JL / Horsley A / Stabat D / Simon M / Johnson S / da Fonseca PCA / Morris EP / Wall JS / Lea SM / Blocker AJ
CitationJournal: Nat Struct Mol Biol / Year: 2009
Title: Three-dimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout.
Authors: 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 /
Abstract: Type III secretion systems (T3SSs) mediate bacterial protein translocation into eukaryotic cells, a process essential for virulence of many Gram-negative pathogens. They are composed of a cytoplasmic ...Type III secretion systems (T3SSs) mediate bacterial protein translocation into eukaryotic cells, a process essential for virulence of many Gram-negative pathogens. They are composed of a cytoplasmic secretion machinery and a base that bridges both bacterial membranes, into which a hollow, external needle is embedded. When isolated, the latter two parts are termed the 'needle complex'. An incomplete understanding of the structure of the needle complex has hampered studies of T3SS function. To estimate the stoichiometry of its components, we measured the mass of its subdomains by scanning transmission electron microscopy (STEM). We determined subunit symmetries by analysis of top and side views within negatively stained samples in low-dose transmission electron microscopy (TEM). Application of 12-fold symmetry allowed generation of a 21-25-A resolution, three-dimensional reconstruction of the needle complex base, revealing many new features and permitting tentative docking of the crystal structure of EscJ, an inner membrane component.
History
DepositionMay 1, 2009-
Header (metadata) releaseMay 20, 2009-
Map releaseMay 27, 2009-
UpdateApr 23, 2010-
Current statusApr 23, 2010Processing site: PDBe / Status: Released

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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
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_1617.map.gz / Format: CCP4 / Size: 22.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a 3D reconstruction of the Shigella flexneri 'needle complex' from negative stain images, done with C12 symmetry imposed. The resolution is 21-25A.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.62 Å/pix.
x 182 pix.
= 476.84 Å
2.62 Å/pix.
x 182 pix.
= 476.84 Å
2.62 Å/pix.
x 182 pix.
= 476.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.62 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1.2
Minimum - Maximum-13.1014 - 9.57075
Average (Standard dev.)-0.00000000106342 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-91-91-91
Dimensions182182182
Spacing182182182
CellA=B=C: 476.84 Å
α=β=γ: 90 °

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
Components
  • Sample: Shigella flexneri T3SS needle complex
  • Protein or peptide: Spa24
  • Protein or peptide: Spa40
  • Protein or peptide: MxiM
  • Protein or peptide: MxiI
  • Protein or peptide: MxiG
  • Protein or peptide: MxiJ
  • Protein or peptide: MxiH
  • Protein or peptide: MxiD

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Supramolecule #1000: Shigella flexneri T3SS needle complex

SupramoleculeName: Shigella flexneri T3SS needle complex / type: sample / ID: 1000
Details: Affinity purified using His6 tag on MxiG N-term Contains detergent (see Zenk et al., 2007)
Oligomeric state: Large macromolecular complex (total list of components not yet known)
Number unique components: 8
Molecular weightExperimental: 3.6 MDa / Method: STEM analysis

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Macromolecule #1: Spa24

MacromoleculeName: Spa24 / type: protein_or_peptide / ID: 1 / Name.synonym: Spa24
Details: This is the MW of the monomer, which must be cleaved in its cytoplasmic portion during T3SS biogenesis.
Oligomeric state: Unknown / Recombinant expression: No
Source (natural)Organism: Shigella flexneri (bacteria) / Strain: Shigella flexneri M90T / synonym: Dysentery bacillus / Cell: Gram-negative bacterium / Organelle: Type III secretion system / Location in cell: inner membrane protein
Molecular weightTheoretical: 240 KDa
SequenceGO: protein secretion / InterPro: Yop virulence translocation protein R

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Macromolecule #2: Spa40

MacromoleculeName: Spa40 / type: protein_or_peptide / ID: 2 / Name.synonym: Spa40
Details: This is the MW of the monomer, which must be cleaved in its cytoplasmic portion during T3SS biogenesis.
Oligomeric state: Unknown / Recombinant expression: No
Source (natural)Organism: Shigella flexneri (bacteria) / Strain: Shigella flexneri M90T / synonym: Dysentery bacillus / Cell: Gram-negative bacterium / Organelle: Type III secretion system / Location in cell: polytopic-inner membrane protein
Molecular weightTheoretical: 400 KDa
SequenceGO: protein secretion
InterPro: Type III exporter system, secretion apparatus protein BsaZ

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Macromolecule #3: MxiM

MacromoleculeName: MxiM / type: protein_or_peptide / ID: 3 / Name.synonym: MxiM / Details: This is the MW of the monomer. / Number of copies: 12 / Recombinant expression: No
Source (natural)Organism: Shigella flexneri (bacteria) / Strain: Shigella flexneri M90T / synonym: Dysentery bacillus / Cell: Gram-negative bacterium / Organelle: Type III secretion system / Location in cell: outside face of outer membrane
Molecular weightTheoretical: 150 KDa
SequenceGO: cell outer membrane

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Macromolecule #4: MxiI

MacromoleculeName: MxiI / type: protein_or_peptide / ID: 4 / Name.synonym: MxiI / Details: This is the MW of the monomer. / Number of copies: 10 / Oligomeric state: Helical polymer / Recombinant expression: No
Source (natural)Organism: Shigella flexneri (bacteria) / Strain: Shigella flexneri M90T / synonym: Dysentery bacillus / Cell: Gram-negative bacterium / Organelle: Type III secretion system / Location in cell: periplasmic space
Molecular weightTheoretical: 100 KDa
SequenceGO: protein transport

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Macromolecule #5: MxiG

MacromoleculeName: MxiG / type: protein_or_peptide / ID: 5 / Name.synonym: MxiG / Details: This is the MW of the monomer. / Number of copies: 24 / Oligomeric state: Probable homo-24mer / Recombinant expression: No
Source (natural)Organism: Shigella flexneri (bacteria) / Strain: Shigella flexneri M90T / synonym: Dysentery bacillus / Cell: Gram-negative bacterium / Organelle: Type III secretion system / Location in cell: spans inner membrane
Molecular weightTheoretical: 430 KDa
SequenceGO: GO: 0009405

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Macromolecule #6: MxiJ

MacromoleculeName: MxiJ / type: protein_or_peptide / ID: 6 / Name.synonym: MxiJ
Details: This is the MW of the monomer with its N-terminal lipid modification site processed (but MW of lipid moiety unknown).
Number of copies: 12 / Oligomeric state: Probable homo-dodecamer / Recombinant expression: No
Source (natural)Organism: Shigella flexneri (bacteria) / Strain: Shigella flexneri M90T / synonym: Dysentery bacillus / Cell: Gram-negative bacterium / Organelle: Type III secretion system / Location in cell: mostly periplasmic face of inner membrane
Molecular weightTheoretical: 250 KDa
SequenceGO: protein secretion / InterPro: Flagellar M-ring , N-terminal

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Macromolecule #7: MxiH

MacromoleculeName: MxiH / type: protein_or_peptide / ID: 7 / Name.synonym: MxiH / Details: This is the MW of the monomer. / Number of copies: 120 / Oligomeric state: helical polymer / Recombinant expression: No
Source (natural)Organism: Shigella flexneri (bacteria) / Strain: Shigella flexneri M90T / synonym: Dysentery bacillus / Cell: Gram-negative bacterium / Organelle: Type III secretion system / Location in cell: Extracellular
Molecular weightTheoretical: 90 KDa
SequenceGO: GO: 0009405 / InterPro: Type III secretion system, needle protein

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Macromolecule #8: MxiD

MacromoleculeName: MxiD / type: protein_or_peptide / ID: 8 / Name.synonym: MxiD
Details: This is the MW of the monomer without it's signal sequence included
Number of copies: 12 / Oligomeric state: homo-dodecamer / Recombinant expression: No
Source (natural)Organism: Shigella flexneri (bacteria) / Strain: Shigella flexneri M90T / synonym: Dysentery bacillus / Cell: Gram-negative bacterium / Organelle: Type III secretion system / Location in cell: Outer membrane
Molecular weightTheoretical: 620 KDa
SequenceGO: protein secretion
InterPro: Type III secretion system outer membrane pore YscC/HrcC

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.050 mg/mL
BufferpH: 8
Details: 10mM Tris pH 8, 0.1% Triton X-100 and 1mM EDTA buffer
StainingType: NEGATIVE
Details: negatively stained in unbuffered 2% aqueous uranyl acetate
GridDetails: 400 mesh copper grids (Athene) covered with holey carbon film over which thin plain carbon was laid were used
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 48600 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 0.9 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 50000
Sample stageSpecimen holder: eucentric / Specimen holder model: OTHER
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 100,000 times magnification
DetailsLow-dose mode was used
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.35 µm / Number real images: 126 / Average electron dose: 20 e/Å2 / Od range: 1 / Bits/pixel: 16

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Image processing

Final two d classificationNumber classes: 4
Final angle assignmentDetails: Imagic beta 87-93 degrees gamma 0-30 degrees
Final reconstructionApplied symmetry - Point group: C12 (12 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: OTHER / Software - Name: SPIDER IMAGIC5
Details: Maps were generated from 41 individual images (selected through image processing out of an initial pool of 3000)
Number images used: 41
DetailsNumbers 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.

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