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
Deposition
Jun 4, 2014
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Header (metadata) release
Jul 2, 2014
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Map release
Jun 17, 2015
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Update
Jun 17, 2015
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Current status
Jun 17, 2015
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Name: 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
Method
cryo EM
Processing
electron tomography
Aggregation state
cell
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Sample preparation
Buffer
pH: 7
Grid
Details: 200 mesh gold grid with thin carbon support, glow discharged in amylamine atmosphere
Vitrification
Cryogen 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
Microscope
FEI POLARA 300
Date
Mar 1, 1999
Image recording
Category: 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 beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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