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- EMDB-0089: Cryo-EM structure of the bacteria-killing type IV secretion syste... -

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Basic information

Entry
Database: EMDB / ID: 0089
TitleCryo-EM structure of the bacteria-killing type IV secretion system core complex from Xanthomonas citriSecretion
Map dataSecretion
SampleCore complex of a bacterial killing type IV secretion system from XanthomonasSecretion:
VirB7 / VirB9 protein / VirB10 protein
Function / homologyType IV secretion system, VirB10 / TraB / TrbI / Conjugal transfer, TrbG/VirB9/CagX / Toxin co-regulated pilus biosynthesis protein Q, C-terminal / VirB9/CagX/TrbG, C-terminal / VirB9/CagX/TrbG, C-terminal domain superfamily / Conjugal transfer protein / Bacterial conjugation TrbI-like protein / Toxin co-regulated pilus biosynthesis protein Q / integral component of membrane / Uncharacterized protein ...Type IV secretion system, VirB10 / TraB / TrbI / Conjugal transfer, TrbG/VirB9/CagX / Toxin co-regulated pilus biosynthesis protein Q, C-terminal / VirB9/CagX/TrbG, C-terminal / VirB9/CagX/TrbG, C-terminal domain superfamily / Conjugal transfer protein / Bacterial conjugation TrbI-like protein / Toxin co-regulated pilus biosynthesis protein Q / integral component of membrane / Uncharacterized protein / VirB9 protein / VirB10 protein
Function and homology information
SourceXanthomonas axonopodis pv. citri str. 306 (bacteria) / Xanthomonas axonopodis pv. citri (strain 306) (bacteria)
Methodsingle particle reconstruction / cryo EM / 3.28 Å resolution
AuthorsCosta TRD / Sgro GG / Farah CS / Waksman G
CitationJournal: Nat Microbiol / Year: 2018
Title: Cryo-EM structure of the bacteria-killing type IV secretion system core complex from Xanthomonas citri.
Authors: Germán G Sgro / Tiago R D Costa / William Cenens / Diorge P Souza / Alexandre Cassago / Luciana Coutinho de Oliveira / Roberto K Salinas / Rodrigo V Portugal / Chuck S Farah / Gabriel Waksman
Validation ReportPDB-ID: 6gyb

SummaryFull reportAbout validation report
DateDeposition: Jun 28, 2018 / Header (metadata) release: Aug 22, 2018 / Map release: Oct 24, 2018 / Last update: Oct 31, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: : PDB-6gyb
  • Surface level: 0.035
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_0089.map.gz (map file in CCP4 format, 171501 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
350 pix
1.06 Å/pix.
= 371. Å
350 pix
1.06 Å/pix.
= 371. Å
350 pix
1.06 Å/pix.
= 371. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density
Contour Level:0.035 (by author), 0.035 (movie #1):
Minimum - Maximum-0.16240412 - 0.27459824
Average (Standard dev.)0.0006112758 (0.008838454)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions350350350
Origin0.00.00.0
Limit349.0349.0349.0
Spacing350350350
CellA=B=C: 370.99997 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z350350350
origin x/y/z0.0000.0000.000
length x/y/z371.000371.000371.000
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS350350350
D min/max/mean-0.1620.2750.001

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Supplemental data

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Sample components

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Entire Core complex of a bacterial killing type IV secretion system from...

EntireName: Core complex of a bacterial killing type IV secretion system from Xanthomonas
Details: Fourteen copies of each of the following three subunits: VirB7, VirB9 and VirB10
Number of components: 4

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Component #1: protein, Core complex of a bacterial killing type IV secretion sy...

ProteinName: Core complex of a bacterial killing type IV secretion system from XanthomonasSecretion
Details: Fourteen copies of each of the following three subunits: VirB7, VirB9 and VirB10
Recombinant expression: No
SourceSpecies: Xanthomonas axonopodis pv. citri str. 306 (bacteria)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #2: protein, VirB7

ProteinName: VirB7 / Number of Copies: 14 / Recombinant expression: No
MassTheoretical: 14.762795 kDa
SourceSpecies: Xanthomonas axonopodis pv. citri (strain 306) (bacteria)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #3: protein, VirB9 protein

ProteinName: VirB9 protein / Number of Copies: 14 / Recombinant expression: No
MassTheoretical: 29.359385 kDa
SourceSpecies: Xanthomonas axonopodis pv. citri (strain 306) (bacteria)
Strain: 306
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #4: protein, VirB10 protein

ProteinName: VirB10 protein / Number of Copies: 14 / Recombinant expression: No
MassTheoretical: 43.392469 kDa
SourceSpecies: Xanthomonas axonopodis pv. citri (strain 306) (bacteria)
Strain: 306
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.3 mg/ml / pH: 8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 295.2 K / Humidity: 100 %
Details: Blot for 4.5 seconds after 30 seconds of incubation..

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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: 6 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 QUANTUM (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1469

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C14 (14 fold cyclic) / Number of projections: 142306
3D reconstructionAlgorithm: BACK PROJECTION / Software: RELION / Resolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Target criteria: Correlation Coefficient / Refinement space: REAL
Details: The electron density was clearly interpretable, which allowed us to build a de novo structural model. This process began by fitting the crystallographic model of the X. citri VirB7 C-terminal N0 domain (PDB:3OV5) and the NMR model of the X. citri VirB9CTD-VirB7NTD complex (PDB:2N01) in order to identify the map with the correct handedness. Models were positioned using Fit in map tool in Chimera, and saved relative to the map. Using these as starting points, we were able to manually build the rest of the model for VirB7 and VirB9CTD, and the de novo models for VirB10CTD, VirB10NTD_150-161 and VirB9NTD using Coot. In this manner, we obtained a combined model for a single VirB7-VirB9-VirB10 heterotrimer unit, which was submitted to iterative rounds of real space refinement and building using PHENIX and Coot software, respectively. Thirteen more copies of the refined heterotrimer were then fit into the density map using Chimera and new rounds of real space refinement (now using NCS for the 42 chains contained in the structure) and building using PHENIX and Coot, respectively, were executed until we obtained good parameters for Ramachandran plot and MolProbity. Chimera and PyMol were used for map and model visualization and figure production.
Overall bvalue: 138
Output model

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