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- EMDB-1422: Structure and composition of the Shigella flexneri "needle comple... -

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Basic information

Entry
Database: EMDB / ID: EMD-1422
TitleStructure and composition of the Shigella flexneri "needle complex", a part of its type III secreton.
Map dataBasal Body of Shigella flexneri T3SS - needle removed
Sample
  • Sample: needle complex or basal body of the Shigella flexneri T3SS
  • Protein or peptide: MxiH
  • Protein or peptide: MxiI
  • Protein or peptide: MxiD
  • Protein or peptide: MxiG
  • Protein or peptide: MxiJ
Function / homology: / protein binding / Type III secretion system outer membrane pore YscC/HrcC / Type III secretion system, needle protein / Flagellar M-ring , N-terminal
Function and homology information
Biological speciesShigella flexneri (bacteria)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 17.0 Å
AuthorsBlocker AJ / Jouihri N / Larquet E / Gounon P / Ebel F / Parsot C / Sansonetti P / Allaoui A
CitationJournal: Mol Microbiol / Year: 2001
Title: Structure and composition of the Shigella flexneri "needle complex", a part of its type III secreton.
Authors: A Blocker / N Jouihri / E Larquet / P Gounon / F Ebel / C Parsot / P Sansonetti / A Allaoui /
Abstract: Type III secretion systems (TTSSs or secretons), essential virulence determinants of many Gram-negative bacteria, serve to translocate proteins directly from the bacteria into the host cytoplasm. ...Type III secretion systems (TTSSs or secretons), essential virulence determinants of many Gram-negative bacteria, serve to translocate proteins directly from the bacteria into the host cytoplasm. Electron microscopy (EM) indicates that the TTSSs of Shigella flexneri are composed of: (1) an external needle; (2) a transmembrane domain; and (3) a cytoplasmic bulb. EM analysis of purified and negatively stained parts 1, 2 and a portion of 3 of the TTSS, together termed the "needle complex" (NC), produced an average image at 17 A resolution in which a base, an outer ring and a needle, inserted through the ring into the base, could be discerned. This analysis and cryoEM images of NCs indicated that the needle and base contain a central 2-3 nm canal. Five major NC components, MxiD, MxiG, MxiJ, MxiH and MxiI, were identified by N-terminal sequencing. MxiG and MxiJ are predicted to be inner membrane proteins and presumably form the base. MxiD is predicted to be an outer membrane protein and to form the outer ring. MxiH and MxiI are small hydrophilic proteins. Mutants lacking either of these proteins formed needleless secretons and were unable to secrete Ipa proteins. As MxiH was present in NCs in large molar excess, we propose that it is the major needle component. MxiI may cap at the external needle tip.
History
DepositionAug 31, 2007-
Header (metadata) releaseSep 12, 2007-
Map releaseSep 12, 2007-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 380
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 380
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_1422.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBasal Body of Shigella flexneri T3SS - needle removed
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
2.2 Å/pix.
x 300 pix.
= 660. Å
2.2 Å/pix.
x 300 pix.
= 660. Å
2.2 Å/pix.
x 300 pix.
= 660. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.2 Å
Density
Contour Level1: 103.0 / Movie #1: 380
Minimum - Maximum-257.841000000000008 - 820.019000000000005
Average (Standard dev.)-12.8026 (±115.599000000000004)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-150-150-149
Dimensions300300300
Spacing300300300
CellA=B=C: 660 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.22.22.2
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z660.000660.000660.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-150-150-149
NX/NY/NZ300300300
MAP C/R/S213
start NC/NR/NS-150-150-149
NC/NR/NS300300300
D min/max/mean-257.841820.019-12.803

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Supplemental data

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Sample components

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Entire : needle complex or basal body of the Shigella flexneri T3SS

EntireName: needle complex or basal body of the Shigella flexneri T3SS
Components
  • Sample: needle complex or basal body of the Shigella flexneri T3SS
  • Protein or peptide: MxiH
  • Protein or peptide: MxiI
  • Protein or peptide: MxiD
  • Protein or peptide: MxiG
  • Protein or peptide: MxiJ

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Supramolecule #1000: needle complex or basal body of the Shigella flexneri T3SS

SupramoleculeName: needle complex or basal body of the Shigella flexneri T3SS
type: sample / ID: 1000 / Oligomeric state: not yet fully determined / Number unique components: 5

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Macromolecule #1: MxiH

MacromoleculeName: MxiH / type: protein_or_peptide / ID: 1 / Details: none; copy number is approximative / Number of copies: 120 / Oligomeric state: helical polymer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Shigella flexneri (bacteria) / Strain: M90T / Tissue: bacterium / Organelle: T3SS / Location in cell: extracellular
Molecular weightExperimental: 9.265 MDa / Theoretical: 9.265 MDa
SequenceInterPro: Type III secretion system, needle protein

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Macromolecule #2: MxiI

MacromoleculeName: MxiI / type: protein_or_peptide / ID: 2 / Details: none; Copy number is approximative / Number of copies: 20 / Oligomeric state: probably helical / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Shigella flexneri (bacteria) / Strain: M90T / Cell: bacterium / Organelle: T3SS / Location in cell: periplasmic
Molecular weightExperimental: 10.633 MDa / Theoretical: 10.633 MDa

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Macromolecule #3: MxiD

MacromoleculeName: MxiD / type: protein_or_peptide / ID: 3
Details: experimental weight is theoretical weight, with predicted signal sequence removed; oligomeric state currently unknown
Oligomeric state: oligomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Shigella flexneri (bacteria) / Strain: M90T / Cell: bacterium / Organelle: T3SS / Location in cell: outer membrane
Molecular weightExperimental: 63.218 MDa / Theoretical: 60.749 MDa
SequenceGO: GO: 0015448
InterPro: Type III secretion system outer membrane pore YscC/HrcC

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Macromolecule #4: MxiG

MacromoleculeName: MxiG / type: protein_or_peptide / ID: 4
Details: no signal sequence cleavage in this protein; oligomeric state unknown
Oligomeric state: oligomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Shigella flexneri (bacteria) / Strain: M90T / Cell: bacterium / Organelle: T3SS / Location in cell: inner membrane
Molecular weightExperimental: 43.002 MDa / Theoretical: 43.002 MDa
SequenceGO: protein binding

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Macromolecule #5: MxiJ

MacromoleculeName: MxiJ / type: protein_or_peptide / ID: 5
Details: experimental weight is theoretical weight, with predicted signal sequence removed; oligomeric state unknown
Oligomeric state: oligomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Shigella flexneri (bacteria) / Strain: M90T / Cell: bacterium / Organelle: T3SS / Location in cell: inner membrane
Molecular weightExperimental: 27.509 MDa / Theoretical: 25.488 MDa
SequenceGO: protein binding / InterPro: Flagellar M-ring , N-terminal

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
Details: samples made in 50 mM Tris pH 8, 5 mM EDTA, 0.1 % v/v Triton X-100 and diluted 1:5 in 20 mM Tris pH 7.4
StainingType: NEGATIVE / Details: 1% uranyl acetate pH 7.5
GridDetails: carbon-coated glow-discharged copper grids
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI/PHILIPS CM120T
Electron beamAcceleration voltage: 100 kV / Electron source: LAB6
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 45000
Sample stageSpecimen holder: Eucentric / Specimen holder model: OTHER
TemperatureMin: 293 K / Max: 293 K / Average: 293 K
Detailslow-dose mode used, imaging done in 1999-2000
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 10 µm / Number real images: 20 / Average electron dose: 10 e/Å2
Details: Hi-Scan rotary drum microdensitometer used, final resolution was 2.2 A/pixel (not 5 A/ pixel as incorrectly stated in Materials and Methods of paper)

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Image processing

Final two d classificationNumber classes: 5
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: OTHER / Software - Name: SPIDER
Details: 1) 2D-average>iterative multireference alignment process (Boekema et al, 1986), using Ward's merging criterion until stable classes obtained; 2) 3D reconstruction> cylindrical symmetry was ...Details: 1) 2D-average>iterative multireference alignment process (Boekema et al, 1986), using Ward's merging criterion until stable classes obtained; 2) 3D reconstruction> cylindrical symmetry was assumed and the final two dimensional projection converted to a three-dimensional map using an iterative back projection procedure (Frank, 1996).
Number images used: 868
DetailsParticles selected using interactive WEB selection program

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