+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2667 | |||||||||
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Title | Intact Shigella Type III secretion injectisomeType three secretion system | |||||||||
Map data | Reconstruction of an intact T3SS machine | |||||||||
Sample |
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Keywords | Secretion machine / T3SS / cytoplasmic complex / injectisome | |||||||||
Biological species | Shigella flexneri 5a (bacteria) | |||||||||
Method | subtomogram averaging / cryo EM / Resolution: 34.0 Å | |||||||||
Authors | Hu B / Margolin W / Rohde J / Picking WL / Picking WD / Liu J | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_2667.map.gz | 13 MB | EMDB map data format | |
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Header (meta data) | emd-2667-v30.xml emd-2667.xml | 8.8 KB 8.8 KB | Display Display | EMDB header |
Images | Shigella-T3SS-WT.jpg | 354.5 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2667 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2667 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_2667.map.gz / Format: CCP4 / Size: 13.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of an intact T3SS machine | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 5 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Shigella flexneri serotype 5a injectisome
Entire | Name: Shigella flexneri serotype 5a injectisome |
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Components |
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-Supramolecule #1000: Shigella flexneri serotype 5a injectisome
Supramolecule | Name: Shigella flexneri serotype 5a injectisome / type: sample / ID: 1000 / Number unique components: 1 |
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-Supramolecule #1: Shigella flexneri serotype 5a injectisome
Supramolecule | Name: 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 |
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Source (natural) | Organism: Shigella flexneri 5a (bacteria) / Strain: Shigella flexneri serotype 5a |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | subtomogram averaging |
Aggregation state | cell |
-Sample preparation
Buffer | pH: 7 |
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Grid | Details: 200 mesh holey carbon grids |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 120 K / Instrument: OTHER / Method: Blot for 3 seconds before plunging |
-Electron microscopy
Microscope | FEI POLARA 300 |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination 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 stage | Specimen holder: Nitrogen cooled / Specimen holder model: SIDE ENTRY, EUCENTRIC / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 ° |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification |
Details | Tilt series were collected in 2x2 binning. |
Date | Jan 1, 2014 |
Image recording | Category: 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 |
-Image processing
Final 3D classification | Number classes: 8 |
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Final reconstruction | Applied 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 |
Details | The subtomograms were visually selected. |