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- PDB-6gyb: Cryo-EM structure of the bacteria-killing type IV secretion syste... -

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Basic information

Entry
Database: PDB / ID: 6gyb
TitleCryo-EM structure of the bacteria-killing type IV secretion system core complex from Xanthomonas citri
Components
  • VirB10 protein
  • VirB7
  • VirB9 protein
KeywordsMEMBRANE PROTEIN / core complex / bacterial killing / protein transport / bacterial Type IV Secretion System
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
Specimen sourceXanthomonas axonopodis pv. citri (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.28 Å resolution
AuthorsSgro, G.G. / Costa, T.R.D. / Farah, C.S. / 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 Report
SummaryFull reportAbout validation report
DateDeposition: Jun 28, 2018 / Release: Oct 24, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Oct 24, 2018Structure modelrepositoryInitial release
1.1Oct 31, 2018Structure modelData collection / Database referencescitation / citation_author_citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-0089
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
a: VirB7
b: VirB9 protein
c: VirB10 protein
d: VirB7
e: VirB9 protein
f: VirB10 protein
g: VirB7
h: VirB9 protein
i: VirB10 protein
j: VirB7
k: VirB9 protein
l: VirB10 protein
m: VirB7
n: VirB9 protein
o: VirB10 protein
p: VirB7
q: VirB9 protein
r: VirB10 protein
s: VirB7
t: VirB9 protein
u: VirB10 protein
A: VirB7
B: VirB9 protein
C: VirB10 protein
D: VirB7
E: VirB9 protein
F: VirB10 protein
G: VirB7
H: VirB9 protein
I: VirB10 protein
J: VirB7
K: VirB9 protein
L: VirB10 protein
M: VirB7
N: VirB9 protein
O: VirB10 protein
P: VirB7
Q: VirB9 protein
R: VirB10 protein
S: VirB7
T: VirB9 protein
U: VirB10 protein


Theoretical massNumber of molelcules
Total (without water)1,225,20542
Polyers1,225,20542
Non-polymers00
Water0
1


  • idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, The core complex of circa 1.13 MDa in size was purified from a HiLoad Superose 6 GL 16/700 gel filtration column.
  • Download structure data
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)211350
ΔGint (kcal/M)-910
Surface area (Å2)308440
MethodPISA

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Components

#1: Protein/peptide
VirB7


Mass: 14762.795 Da / Num. of mol.: 14
Source: (gene. exp.) Xanthomonas axonopodis pv. citri (strain 306) (bacteria)
Gene: XAC2622 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8PJB3
#2: Protein/peptide
VirB9 protein


Mass: 29359.385 Da / Num. of mol.: 14
Source: (gene. exp.) Xanthomonas axonopodis pv. citri (strain 306) (bacteria)
Strain: 306 / Gene: virB9, XAC2620 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8PJB5
#3: Protein/peptide
VirB10 protein


Mass: 43392.469 Da / Num. of mol.: 14
Source: (gene. exp.) Xanthomonas axonopodis pv. citri (strain 306) (bacteria)
Strain: 306 / Gene: virB10, XAC2619 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8PJB6

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Core complex of a bacterial killing type IV secretion system from XanthomonasSecretion
Type: COMPLEX
Details: Fourteen copies of each of the following three subunits: VirB7, VirB9 and VirB10
Entity ID: 1, 2, 3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Xanthomonas axonopodis pv. citri str. 306 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer ID
120 mMTris-HClNH2C(CH2OH)3HCl1
2200 mMSodium ChlorideNaCl1
310 mMLDAOCH3(CH2)11N(O)(CH3)21
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295.2 kelvins
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
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingAverage exposure time: 12 sec. / Electron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Number of grids imaged: 1 / Number of real images: 1469
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
1RELION2.0particle selection
2EPU1.8image acquisition
4Gctf1.06CTF correction
7Coot0.8.6model fitting
8UCSF Chimera8.6.1model fitting
10cryoSPARC1.0initial Euler assignment
11RELION2.0final Euler assignment
12RELION2.0classification
13RELION2.03D reconstruction
14PHENIX1.12model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 185079
SymmetryPoint symmetry: C14
3D reconstructionResolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 142306 / Algorithm: BACK PROJECTION / Number of class averages: 1 / Symmetry type: POINT
Atomic model buildingDetails: 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 b value: 138 / Ref space: REAL / Target criteria: Correlation Coefficient
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00860690
ELECTRON MICROSCOPYf_angle_d0.89282586
ELECTRON MICROSCOPYf_dihedral_angle_d10.32436232
ELECTRON MICROSCOPYf_chiral_restr0.0609338
ELECTRON MICROSCOPYf_plane_restr0.00710570

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