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- EMDB-2480: Tomogram of Injectisome (wild type) from Salmonella typhiumurium ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2480
TitleTomogram of Injectisome (wild type) from Salmonella typhiumurium (substrate trapped) in situ
Map dataTomogram of substrate trapped type-3 secretion systems from Salmonella typhimurium
Sample
  • Sample: Type-3 secretion system from Salmonella typhimurium in situ (substrate trapped): Visualization of unfolded protein transport across membranes
  • Organelle or cellular component: type 3 secretion systemType three secretion system
KeywordsInjectisome / Pathogenic Type-3 Secretion System / Protein delivery machine / Salmonella typhimurium / cryo electron tomography
Biological speciesSalmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Methodelectron tomography / cryo EM
AuthorsRadics J / Konigsmaier L / Marlovits TC
CitationJournal: Nat Struct Mol Biol / Year: 2014
Title: Structure of a pathogenic type 3 secretion system in action.
Authors: Julia Radics / Lisa Königsmaier / Thomas C Marlovits /
Abstract: Type 3 secretion systems use 3.5-megadalton syringe-like, membrane-embedded 'injectisomes', each containing an ~800-Å-long needle complex to connect intracellular compartments of infectious bacteria ...Type 3 secretion systems use 3.5-megadalton syringe-like, membrane-embedded 'injectisomes', each containing an ~800-Å-long needle complex to connect intracellular compartments of infectious bacteria and hosts. Here we identify requirements for substrate association with, transport through and exit from the injectisome of Salmonella enterica serovar Typhimurium. This guided the design of substrates that become trapped within the secretion path and enabled visualization of injectisomes in action in situ. We used cryo-EM to define the secretion path, providing a structural explanation as to why effector proteins must be unfolded during transport. Furthermore, trapping of a heterologous substrate in the needle prevents secretion of natural bacterial effectors. Together, the data reveal the path of protein secretion across multiple membranes and show that mechanisms rejecting unacceptable substrates can be undermined, and transport of bacterial effectors across an already assembled type 3 secretion system can be inhibited.
History
DepositionSep 27, 2013-
Header (metadata) releaseNov 13, 2013-
Map releaseDec 11, 2013-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: PDBe / Status: Released

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Structure visualization

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Supplemental images

emd_2480.tif

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Map

FileDownload / File: emd_2480.map.gz / Format: CCP4 / Size: 52.2 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationTomogram of substrate trapped type-3 secretion systems from Salmonella typhimurium
Voxel sizeX=Y=Z: 19.88 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)-64.995986939999995 (±3.70534849)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-1-30247
Dimensions768722101
Spacing768722101
CellA: 14353.359 Å / B: 15267.84 Å / C: 2007.8799 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z19.87999861495819.8819.88
M x/y/z722768101
origin x/y/z0.0000.0000.000
length x/y/z14353.35915267.8402007.880
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS-302-147
NC/NR/NS722768101
D min/max/mean-128.000127.000-64.996

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Supplemental data

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Sample components

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Entire : Type-3 secretion system from Salmonella typhimurium in situ (subs...

EntireName: Type-3 secretion system from Salmonella typhimurium in situ (substrate trapped): Visualization of unfolded protein transport across membranes
Components
  • Sample: Type-3 secretion system from Salmonella typhimurium in situ (substrate trapped): Visualization of unfolded protein transport across membranes
  • Organelle or cellular component: type 3 secretion systemType three secretion system

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Supramolecule #1000: Type-3 secretion system from Salmonella typhimurium in situ (subs...

SupramoleculeName: Type-3 secretion system from Salmonella typhimurium in situ (substrate trapped): Visualization of unfolded protein transport across membranes
type: sample / ID: 1000 / Oligomeric state: 1 / Number unique components: 1
Molecular weightTheoretical: 3.5 MDa

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Supramolecule #1: type 3 secretion system

SupramoleculeName: type 3 secretion system / type: organelle_or_cellular_component / ID: 1 / Name.synonym: injectisome / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Strain: SB905 / Location in cell: Plasma membrane
Molecular weightTheoretical: 3.5 MDa

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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: 8 / Details: 10 mM Tris, 5 mM EDTA
GridDetails: 400 mesh Mo-Grid Quantifoil R2/1
VitrificationCryogen name: ETHANE / Chamber humidity: 65 % / Chamber temperature: 91 K / Instrument: LEICA EM GP
Method: 1. grids glowdischarged (300sec/20mAmp) 2. sample 5ul applied for 5sec (cell settling) 3. blotting for 2sec/distance 189

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 30120 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 9.0 µm / Nominal magnification: 23000
Sample stageSpecimen holder: liquid nitrogen cooled / Specimen holder model: GATAN HELIUM / Tilt series - Axis1 - Max angle: 60 ° / Tilt series - Axis1 - Angle increment: 2 °
TemperatureMin: 83 K / Max: 108 K / Average: 93 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at appr 200.000 magnification
DateMar 2, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 60 / Average electron dose: 100 e/Å2 / Details: Image acquisition using SerialEM
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: OTHER / Software - Name: IMOD / Number images used: 60
DetailsIMOD (weighted back-projection)

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