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- PDB-6nym: Helicobacter pylori Vacuolating Cytotoxin A Oligomeric Assembly 2... -

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Basic information

Entry
Database: PDB / ID: 6nym
TitleHelicobacter pylori Vacuolating Cytotoxin A Oligomeric Assembly 2d (OA-2d)
ComponentsVacuolating cytotoxin autotransporter
KeywordsTOXIN / Helicobacter pylori / vacuolating cytotoxin A / pore-forming toxin
Function / homology
Function and homology information


cell outer membrane / toxin activity / periplasmic space / cell surface / extracellular region
Similarity search - Function
Vacuolating cytotoxin / Vacuolating cyotoxin / Outer membrane autotransporter barrel / Autotransporter beta-domain / Autotransporter beta-domain profile. / Autotransporter beta-domain / Autotransporter beta-domain superfamily
Similarity search - Domain/homology
Vacuolating cytotoxin autotransporter
Similarity search - Component
Biological speciesHelicobacter pylori (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsZhang, K. / Zhang, H. / Li, S. / Au, S. / Chiu, W.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)P41GM103832 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R01GM079429 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)S10OD021600 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: Cryo-EM structures of vacuolating cytotoxin A oligomeric assemblies at near-atomic resolution.
Authors: Kaiming Zhang / Huawei Zhang / Shanshan Li / Grigore D Pintilie / Tung-Chung Mou / Yuanzhu Gao / Qinfen Zhang / Henry van den Bedem / Michael F Schmid / Shannon Wing Ngor Au / Wah Chiu /
Abstract: Human gastric pathogen () is the primary risk factor for gastric cancer and is one of the most prevalent carcinogenic infectious agents. Vacuolating cytotoxin A (VacA) is a key virulence factor ...Human gastric pathogen () is the primary risk factor for gastric cancer and is one of the most prevalent carcinogenic infectious agents. Vacuolating cytotoxin A (VacA) is a key virulence factor secreted by and induces multiple cellular responses. Although structural and functional studies of VacA have been extensively performed, the high-resolution structure of a full-length VacA protomer and the molecular basis of its oligomerization are still unknown. Here, we use cryoelectron microscopy to resolve 10 structures of VacA assemblies, including monolayer (hexamer and heptamer) and bilayer (dodecamer, tridecamer, and tetradecamer) oligomers. The models of the 88-kDa full-length VacA protomer derived from the near-atomic resolution maps are highly conserved among different oligomers and show a continuous right-handed β-helix made up of two domains with extensive domain-domain interactions. The specific interactions between adjacent protomers in the same layer stabilizing the oligomers are well resolved. For double-layer oligomers, we found short- and/or long-range hydrophobic interactions between protomers across the two layers. Our structures and other previous observations lead to a mechanistic model wherein VacA hexamer would correspond to the prepore-forming state, and the N-terminal region of VacA responsible for the membrane insertion would undergo a large conformational change to bring the hydrophobic transmembrane region to the center of the oligomer for the membrane channel formation.
History
DepositionFeb 11, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 27, 2019Provider: repository / Type: Initial release
Revision 1.1Apr 3, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.pdbx_database_id_PubMed ..._citation.journal_abbrev / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Apr 17, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
A: Vacuolating cytotoxin autotransporter
B: Vacuolating cytotoxin autotransporter
C: Vacuolating cytotoxin autotransporter
D: Vacuolating cytotoxin autotransporter
E: Vacuolating cytotoxin autotransporter
F: Vacuolating cytotoxin autotransporter
G: Vacuolating cytotoxin autotransporter
H: Vacuolating cytotoxin autotransporter
I: Vacuolating cytotoxin autotransporter
J: Vacuolating cytotoxin autotransporter
K: Vacuolating cytotoxin autotransporter
L: Vacuolating cytotoxin autotransporter


Theoretical massNumber of molelcules
Total (without water)1,061,55912
Polymers1,061,55912
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area25290 Å2
ΔGint-90 kcal/mol
Surface area349810 Å2

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Components

#1: Protein
Vacuolating cytotoxin autotransporter


Mass: 88463.211 Da / Num. of mol.: 12 / Source method: isolated from a natural source / Source: (natural) Helicobacter pylori (bacteria) / References: UniProt: Q48245

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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: Helicobacter pylori Vacuolating Cytotoxin A Oligomeric Assembly 2d (OA-2d)
Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightValue: 0.088 MDa / Experimental value: YES
Source (natural)Organism: Helicobacter pylori (bacteria) / Strain: 60190
Buffer solutionpH: 7.4
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: VacA OA-2d
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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 / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1300 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 7 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 13708
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 30 / Used frames/image: 1-30

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategory
1EMAN22.2particle selection
2EPUimage acquisition
4CTFFIND4CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10RELION2.1initial Euler assignment
11cryoSPARC2final Euler assignment
12RELION2classification
13cryoSPARC23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 540214
SymmetryPoint symmetry: D6 (2x6 fold dihedral)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 31625 / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00569888
ELECTRON MICROSCOPYf_angle_d0.86994656
ELECTRON MICROSCOPYf_dihedral_angle_d4.82641136
ELECTRON MICROSCOPYf_chiral_restr0.05810620
ELECTRON MICROSCOPYf_plane_restr0.00512396

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