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- EMDB-2667: Intact Shigella Type III secretion injectisome -

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Basic information

Entry
Database: EMDB / ID: EMD-2667
TitleIntact Shigella Type III secretion injectisomeType three secretion system
Map dataReconstruction of an intact T3SS machine
Sample
  • Sample: Shigella flexneri serotype 5a injectisome
  • Organelle or cellular component: Shigella flexneri serotype 5a injectisome
KeywordsSecretion machine / T3SS / cytoplasmic complex / injectisome
Biological speciesShigella flexneri 5a (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 34.0 Å
AuthorsHu B / Margolin W / Rohde J / Picking WL / Picking WD / Liu J
CitationJournal: Proc Natl Acad Sci U S A / Year: 2015
Title: Visualization of the type III secretion sorting platform of Shigella flexneri.
Authors: Bo Hu / Dustin R Morado / William Margolin / John R Rohde / Olivia Arizmendi / Wendy L Picking / William D Picking / Jun Liu /
Abstract: Bacterial type III secretion machines are widely used to inject virulence proteins into eukaryotic host cells. These secretion machines are evolutionarily related to bacterial flagella and consist of ...Bacterial type III secretion machines are widely used to inject virulence proteins into eukaryotic host cells. These secretion machines are evolutionarily related to bacterial flagella and consist of a large cytoplasmic complex, a transmembrane basal body, and an extracellular needle. The cytoplasmic complex forms a sorting platform essential for effector selection and needle assembly, but it remains largely uncharacterized. Here we use high-throughput cryoelectron tomography (cryo-ET) to visualize intact machines in a virulent Shigella flexneri strain genetically modified to produce minicells capable of interaction with host cells. A high-resolution in situ structure of the intact machine determined by subtomogram averaging reveals the cytoplasmic sorting platform, which consists of a central hub and six spokes, with a pod-like structure at the terminus of each spoke. Molecular modeling of wild-type and mutant machines allowed us to propose a model of the sorting platform in which the hub consists mainly of a hexamer of the Spa47 ATPase, whereas the MxiN protein comprises the spokes and the Spa33 protein forms the pods. Multiple contacts among those components are essential to align the Spa47 ATPase with the central channel of the MxiA protein export gate to form a unique nanomachine. The molecular architecture of the Shigella type III secretion machine and its sorting platform provide the structural foundation for further dissecting the mechanisms underlying type III secretion and pathogenesis and also highlight the major structural distinctions from bacterial flagella.
History
DepositionJun 4, 2014-
Header (metadata) releaseJul 2, 2014-
Map releaseJun 17, 2015-
UpdateJun 17, 2015-
Current statusJun 17, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Shigella-T3S...

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Map

FileDownload / File: emd_2667.map.gz / Format: CCP4 / Size: 13.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of an intact T3SS machine
Voxel sizeX=Y=Z: 5 Å
Density
Contour LevelBy AUTHOR: 0.2 / Movie #1: 0.2
Minimum - Maximum-5.61434698 - 7.70953178
Average (Standard dev.)0.0 (±0.99999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-60-60-130
Dimensions120120260
Spacing120120260
CellA: 600.0 Å / B: 600.0 Å / C: 1300.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z555
M x/y/z120120260
origin x/y/z0.0000.0000.000
length x/y/z600.000600.0001300.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-24-24-24
NX/NY/NZ494949
MAP C/R/S123
start NC/NR/NS-60-60-130
NC/NR/NS120120260
D min/max/mean-5.6147.7100.000

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Supplemental data

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Sample components

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Entire : Shigella flexneri serotype 5a injectisome

EntireName: Shigella flexneri serotype 5a injectisome
Components
  • Sample: Shigella flexneri serotype 5a injectisome
  • Organelle or cellular component: Shigella flexneri serotype 5a injectisome

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Supramolecule #1000: Shigella flexneri serotype 5a injectisome

SupramoleculeName: Shigella flexneri serotype 5a injectisome / type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: Shigella flexneri serotype 5a injectisome

SupramoleculeName: Shigella flexneri serotype 5a injectisome / type: organelle_or_cellular_component / ID: 1
Details: Minicell derived from Shigella flexneri serotype 5a
Number of copies: 25 / Recombinant expression: No
Source (natural)Organism: Shigella flexneri 5a (bacteria) / Strain: Shigella flexneri serotype 5a

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7
GridDetails: 200 mesh holey carbon grids
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 120 K / Instrument: OTHER / Method: Blot for 3 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 8.0 µm / Nominal defocus min: 5.0 µm / Nominal magnification: 15500
Sample stageSpecimen holder: Nitrogen cooled / Specimen holder model: SIDE ENTRY, EUCENTRIC / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DetailsTilt series were collected in 2x2 binning.
DateJan 1, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 28548 / Average electron dose: 60 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Final 3D classificationNumber classes: 8
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 34.0 Å / Resolution method: OTHER / Software - Name: IMOD, RAPTOR, PROTOMO / Number subtomograms used: 1448
DetailsThe subtomograms were visually selected.

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