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- EMDB-2805: Refined negative stain electron microscopy reconstruction of the ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2805
TitleRefined negative stain electron microscopy reconstruction of the tip complex from the type III secretion system of Shigella flexneri with MxiH mutation Q51A
Map dataReconstruction of the tip complex and needle from the type III secretion system of Shigella flexneri with MxiH mutation Q51A and using groups of individual images
Sample
  • Sample: Conformation of the tip complex from the type III secretion system of Shigella flexneri bound to the needle with MxiH mutation Q51A
  • Organelle or cellular component: Tip complex and needle
  • Organelle or cellular component: Tip complex and needle
KeywordsTip complex / Type III secretion system / Shigella flexneri / Refined Q51A
Biological speciesShigella flexneri (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 19.0 Å
AuthorsCheung M / Shen D / Makino F / Kato T / Roehrich D / Martinez-Argudo I / Walker ML / Murillo I / Liu X / Pain M ...Cheung M / Shen D / Makino F / Kato T / Roehrich D / Martinez-Argudo I / Walker ML / Murillo I / Liu X / Pain M / Brown J / Frazer G / Mantell J / Mina P / Todd T / Sessions RB / Namba K / Blocker AJ
CitationJournal: Proc Natl Acad Sci U S A / Year: 2008
Title: What's the point of the type III secretion system needle?
Authors: Ariel J Blocker / Janet E Deane / Andreas K J Veenendaal / Pietro Roversi / Julie L Hodgkinson / Steven Johnson / Susan M Lea /
Abstract: Recent work by several groups has significantly expanded our knowledge of the structure, regulation of assembly, and function of components of the extracellular portion of the type III secretion ...Recent work by several groups has significantly expanded our knowledge of the structure, regulation of assembly, and function of components of the extracellular portion of the type III secretion system (T3SS) of Gram-negative bacteria. This perspective presents a structure-informed analysis of functional data and discusses three nonmutually exclusive models of how a key aspect of T3SS biology, the sensing of host cells, may be performed.
History
DepositionOct 17, 2014-
Header (metadata) releaseNov 5, 2014-
Map releaseDec 3, 2014-
UpdateMar 11, 2015-
Current statusMar 11, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0361
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0361
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0361
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2805.png

Downloads & links

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Map

FileDownload / File: emd_2805.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the tip complex and needle from the type III secretion system of Shigella flexneri with MxiH mutation Q51A and using groups of individual images
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.12 Å/pix.
x 200 pix.
= 424. Å		2.12 Å/pix.
x 200 pix.
= 424. Å		2.12 Å/pix.
x 200 pix.
= 424. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.12 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0361 / Movie #1: 0.0361
Minimum - Maximum-0.06119302 - 0.10173064
Average (Standard dev.)0.00004494 (±0.00715851)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 423.99997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.122.122.12
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z424.000424.000424.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-40-32-96
NX/NY/NZ8165193
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0610.1020.000

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Supplemental data

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Sample components

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Entire : Conformation of the tip complex from the type III secretion syste...

EntireName: Conformation of the tip complex from the type III secretion system of Shigella flexneri bound to the needle with MxiH mutation Q51A
Components
  • Sample: Conformation of the tip complex from the type III secretion system of Shigella flexneri bound to the needle with MxiH mutation Q51A
  • Organelle or cellular component: Tip complex and needle
  • Organelle or cellular component: Tip complex and needle

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Supramolecule #1000: Conformation of the tip complex from the type III secretion syste...

SupramoleculeName: Conformation of the tip complex from the type III secretion system of Shigella flexneri bound to the needle with MxiH mutation Q51A
type: sample / ID: 1000
Details: Tip complexes were analysed from purified needle complexes and therefore when still bound to the needle tip
Oligomeric state: Five subunits of the tip complex bound to fifty subunits of the needle
Number unique components: 2
Molecular weightTheoretical: 680 KDa

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Supramolecule #1: Tip complex and needle

SupramoleculeName: Tip complex and needle / type: organelle_or_cellular_component / ID: 1 / Number of copies: 1 / Oligomeric state: Heteropentameric / Recombinant expression: Yes
Ref GO0: GO:0030257
Ref INTERPRO0: IPR006135
Source (natural)Organism: Shigella flexneri (bacteria) / Strain: serotype 5a / Organelle: Type III secretion system
Location in cell: Extracellular component of membrane embedded complex
Molecular weightTheoretical: 210 KDa
Recombinant expressionOrganism: Shigella flexneri (bacteria) / Recombinant strain: M90T derivative / Recombinant plasmid: pACT3, pBAD

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Supramolecule #2: Tip complex and needle

SupramoleculeName: Tip complex and needle / type: organelle_or_cellular_component / ID: 2 / Number of copies: 1 / Recombinant expression: Yes
Ref GO0: GO:0030257
Ref INTERPRO0: IPR006135
Source (natural)Organism: Shigella flexneri (bacteria) / Strain: serotype 5a / Organelle: Type III secretion system
Location in cell: Extracellular component of membrane embedded complex
Molecular weightTheoretical: 470 KDa
Recombinant expressionOrganism: Shigella flexneri (bacteria) / Recombinant strain: M90T derivative / Recombinant plasmid: pACT3, pBAD

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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

Concentration0.1 mg/mL
BufferpH: 8
Details: 0.1% (w/v) DDM, 150 mM NaCl, 25 mM Tris pH 8, 5 mM EDTA
StainingType: NEGATIVE
Details: Carbon coating mica sheets were submerged in a drop of 0.01% (w/v) DDM until the carbon sheet partially detached from the mica. The mica was then submerged in the sample for 5 s, then ...Details: Carbon coating mica sheets were submerged in a drop of 0.01% (w/v) DDM until the carbon sheet partially detached from the mica. The mica was then submerged in the sample for 5 s, then submerged in a drop of water for 5 s. The mica was then placed onto the surface of a drop of 1% uranyl acetate and allowed to sink, causing the carbon to float on the surface of the drop. After 10 s, the carbon sheet was adsorbed onto a grid.
GridDetails: 300 mesh copper grid with a thin carbon film to which the specimen was adsorbed to
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 20
Alignment procedureLegacy - Astigmatism: Thon rings visualised at 50-100,000 times magnification using the fast FT and corrected accordingly
Legacy - Electron beam tilt params: 0
DateAug 31, 2011
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 250 / Average electron dose: 9 e/Å2 / Camera length: 460 / Od range: 3.56 / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 70754 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 1.4 µm / Nominal defocus min: 1.4 µm / Nominal magnification: 50000
Sample stageSpecimen holder: FEI single tilt holder / Specimen holder model: SIDE ENTRY, EUCENTRIC

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Image processing

DetailsParticles were selected using helix boxer from EMAN2. After alignment, Euler angles were determined by projection matching with projections of a map Q51A. The reconstruction was iterated and helical pattern searching and imposition was applied to each new reference generated after each iteration.
CTF correctionDetails: First zero set to 18 angstrom, no CTF correction not required
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: OTHER
Software - Name: EMAN2, SPIDER, bespoke-2D-alignment, bespoke-X-and-Y-shifting
Number images used: 2545
Final angle assignmentDetails: SPIDER: theta 4 degrees

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