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- EMDB-2669: Spa33 mutant structure of Shigella Type III secretion injectisome -

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Basic information

Entry
Database: EMDB / ID: EMD-2669
TitleSpa33 mutant structure of Shigella Type III secretion injectisome
Map dataSpa33 mutant structure
Sample
  • Sample: Spa33 mutant T3SS structure
  • Organelle or cellular component: Shigella flexneri
KeywordsSpa33 mutant / T3SS / Secretion
Biological speciesShigella flexneri (bacteria)
Methodelectron tomography / cryo EM
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: 0.6
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2669.png

Downloads & links

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Map

FileDownload / File: emd_2669.map.gz / Format: CCP4 / Size: 955.1 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSpa33 mutant structure
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
10 Å/pix.
x 100 pix.
= 1000. Å		10 Å/pix.
x 50 pix.
= 500. Å		10 Å/pix.
x 50 pix.
= 500. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 10 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.6 / Movie #1: 0.6
Minimum - Maximum-3.69838858 - 4.57369328
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-25-25-50
Dimensions5050100
Spacing5050100
CellA: 500.0 Å / B: 500.0 Å / C: 1000.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z101010
M x/y/z5050100
origin x/y/z0.0000.0000.000
length x/y/z500.000500.0001000.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-24-24-24
NX/NY/NZ494949
MAP C/R/S123
start NC/NR/NS-25-25-50
NC/NR/NS5050100
D min/max/mean-3.6984.5740.000

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Supplemental data

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Sample components

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Entire : Spa33 mutant T3SS structure

EntireName: Spa33 mutant T3SS structure
Components
  • Sample: Spa33 mutant T3SS structure
  • Organelle or cellular component: Shigella flexneri

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Supramolecule #1000: Spa33 mutant T3SS structure

SupramoleculeName: Spa33 mutant T3SS structure / type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: Shigella flexneri

SupramoleculeName: Shigella flexneri / type: organelle_or_cellular_component / ID: 1 / Number of copies: 20 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Shigella flexneri (bacteria)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7
GridDetails: 200 mesh gold grid with thin carbon support, glow discharged in amylamine atmosphere
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 120 K / Instrument: OTHER
Timed resolved state: e.g. Vitrified 30 msec after spraying with effector
Method: Blot for 3 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
DateMar 1, 1999
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 27023 / Details: 27023 / Bits/pixel: 16
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 15500
Sample stageSpecimen holder model: OTHER / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 ° / Tilt series - Axis1 - Angle increment: 2 °
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsCTF correction was not performed on each projection
Final reconstructionAlgorithm: OTHER / Resolution method: OTHER / Number images used: 61

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