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- EMDB-8399: Single particle cryo-EM reconstruction of the Salmonella SPI-1 ty... -

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Entry
Database: EMDB / ID: EMD-8399
TitleSingle particle cryo-EM reconstruction of the Salmonella SPI-1 type III secretion injectisome secretin InvG at 3.6 Angstrom resolutionType three secretion system
Map dataType three secretion system
SampleType III injectisome basal body:
Protein InvG
Function / homology
Function and homology information


type III protein secretion system complex / protein secretion / cell outer membrane / pathogenesis / identical protein binding
Type II secretion system protein GspD, conserved site / Type III secretion system outer membrane pore YscC/HrcC / Type II/III secretion system / NolW-like / NolW-like superfamily
Type 3 secretion system secretin
Biological speciesSalmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsWorrall LJ / Hong C / Vuckovic M / Bergeron JRC / Huang RK / Yu Z / Strynadka NCJ
Funding support Canada, 1 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
CitationJournal: Nature / Year: 2016
Title: Near-atomic-resolution cryo-EM analysis of the Salmonella T3S injectisome basal body.
Authors: L J Worrall / C Hong / M Vuckovic / W Deng / J R C Bergeron / D D Majewski / R K Huang / T Spreter / B B Finlay / Z Yu / N C J Strynadka /
Abstract: The type III secretion (T3S) injectisome is a specialized protein nanomachine that is critical for the pathogenicity of many Gram-negative bacteria, including purveyors of plague, typhoid fever, ...The type III secretion (T3S) injectisome is a specialized protein nanomachine that is critical for the pathogenicity of many Gram-negative bacteria, including purveyors of plague, typhoid fever, whooping cough, sexually transmitted infections and major nosocomial infections. This syringe-shaped 3.5-MDa macromolecular assembly spans both bacterial membranes and that of the infected host cell. The internal channel formed by the injectisome allows for the direct delivery of partially unfolded virulence effectors into the host cytoplasm. The structural foundation of the injectisome is the basal body, a molecular lock-nut structure composed predominantly of three proteins that form highly oligomerized concentric rings spanning the inner and outer membranes. Here we present the structure of the prototypical Salmonella enterica serovar Typhimurium pathogenicity island 1 basal body, determined using single-particle cryo-electron microscopy, with the inner-membrane-ring and outer-membrane-ring oligomers defined at 4.3 Å and 3.6 Å resolution, respectively. This work presents the first, to our knowledge, high-resolution structural characterization of the major components of the basal body in the assembled state, including that of the widespread class of outer-membrane portals known as secretins.
Validation ReportPDB-ID: 5tcq

SummaryFull reportAbout validation report
History
DepositionSep 15, 2016-
Header (metadata) releaseNov 9, 2016-
Map releaseDec 21, 2016-
UpdateJan 15, 2020-
Current statusJan 15, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5tcq
  • Surface level: 0.08
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-5tcq
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8399.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.71 Å/pix.
x 300 pix.
= 513. Å
1.71 Å/pix.
x 300 pix.
= 513. Å
1.71 Å/pix.
x 300 pix.
= 513. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.71 Å
Density
Contour LevelBy AUTHOR: 0.08 / Movie #1: 0.08
Minimum - Maximum-0.18196891 - 0.3536772
Average (Standard dev.)0.0008786313 (±0.0072768456)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 513.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.711.711.71
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z513.000513.000513.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-152-37
NX/NY/NZ998271
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.1820.3540.001

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Supplemental data

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Sample components

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Entire Type III injectisome basal body

EntireName: Type III injectisome basal body / Details: PrgH130-392 mutant / Number of components: 2

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Component #1: protein, Type III injectisome basal body

ProteinName: Type III injectisome basal body / Details: PrgH130-392 mutant / Recombinant expression: No
MassTheoretical: 2 MDa
SourceSpecies: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)

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Component #2: protein, Protein InvG

ProteinName: Protein InvG / Number of Copies: 15 / Recombinant expression: No
MassTheoretical: 61.835559 kDa
SourceSpecies: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 10 mg/mL / pH: 8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 298 K / Humidity: 90 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.3 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 64000.0 X (nominal), 29240.0 X (calibrated) / Cs: 0.01 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500.0 - 3000.0 nm / Energy filter: Gatan GIF / Energy window: 0-20 eV
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: (80.0 - 80.0 K)
CameraDetector: GATAN K2 QUANTUM (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 2685 / Sampling size: 5 µm

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C15 (15 fold cyclic) / Number of projections: 83900
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Refinement space: REAL
Details: Initial fitting was carried out with Chimera, followed by rebuilding and refinement in Rosetta, Phenix, and Coot.
Output model

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