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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 / homology
Function and homology information


membrane => GO:0016020
Similarity search - Function
Toxin co-regulated pilus biosynthesis protein Q, C-terminal / Toxin co-regulated pilus biosynthesis protein Q / Conjugal transfer, TrbG/VirB9/CagX / VirB9/CagX/TrbG, C-terminal / VirB9/CagX/TrbG, C-terminal domain superfamily / Conjugal transfer protein / Type IV secretion system, VirB10/TrbI / Bacterial conjugation TrbI-like protein / Type IV secretion system, VirB10 / TraB / TrbI
Similarity search - Domain/homology
Toxin co-regulated pilus biosynthesis protein Q C-terminal domain-containing protein / VirB9 protein / VirB10 protein
Similarity search - Component
Biological speciesXanthomonas axonopodis pv. citri (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsSgro, G.G. / Costa, T.R.D. / Farah, C.S. / Waksman, G.
Funding support United Kingdom, Brazil, 3items
OrganizationGrant numberCountry
Wellcome Trust098302 United Kingdom
Sao Paulo Research Foundation2011/07777-5 Brazil
Sao Paulo Research Foundation2017/17303-7 Brazil
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 /
Abstract: Type IV secretion (T4S) systems form the most common and versatile class of secretion systems in bacteria, capable of injecting both proteins and DNAs into host cells. T4S systems are typically ...Type IV secretion (T4S) systems form the most common and versatile class of secretion systems in bacteria, capable of injecting both proteins and DNAs into host cells. T4S systems are typically composed of 12 components that form 2 major assemblies: the inner membrane complex embedded in the inner membrane and the core complex embedded in both the inner and outer membranes. Here we present the 3.3 Å-resolution cryo-electron microscopy model of the T4S system core complex from Xanthomonas citri, a phytopathogen that utilizes this system to kill bacterial competitors. An extensive mutational investigation was performed to probe the vast network of protein-protein interactions in this 1.13-MDa assembly. This structure expands our knowledge of the molecular details of T4S system organization, assembly and evolution.
History
DepositionJun 28, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 24, 2018Provider: repository / Type: Initial release
Revision 1.1Oct 31, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Dec 5, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Nov 6, 2019Group: Data collection / Refinement description / Category: em_3d_fitting / Item: _em_3d_fitting.target_criteria
Revision 1.4Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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  • 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:
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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
Polymers1,225,20542
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.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area211350 Å2
ΔGint-910 kcal/mol
Surface area308440 Å2
MethodPISA

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Components

#1: Protein
VirB7


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


Mass: 29359.385 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
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
VirB10 protein


Mass: 43392.469 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
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 / 3D 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: all / 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-HClTrisNH2C(CH2OH)3HCl1
2200 mMSodium ChlorideNaClSodium chloride1
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 K
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
MicroscopyModel: 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) / Num. of grids imaged: 1 / Num. 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
1RELION2particle selection
2EPU1.8image acquisition
4Gctf1.06CTF correction
7Coot0.8.6model fitting
8UCSF Chimera8.6.1model fitting
10cryoSPARC1initial Euler assignment
11RELION2final Euler assignment
12RELION2classification
13RELION23D reconstruction
14PHENIX1.12model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 185079
SymmetryPoint symmetry: C14 (14 fold cyclic)
3D reconstructionResolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 142306 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 138 / Space: REAL / Target criteria: Cross-correlation coefficient
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- ...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.
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_restr0.069338
ELECTRON MICROSCOPYf_plane_restr0.00710570

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