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- EMDB-0548: Helicobacter pylori Vacuolating Cytotoxin A Oligomeric Assembly 2... -

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Basic information

Entry
Database: EMDB / ID: EMD-0548
TitleHelicobacter pylori Vacuolating Cytotoxin A Oligomeric Assembly 2f (OA-2f)
Map dataHelicobacter pylori Vacuolating Cytotoxin A Oligomeric Assembly 2f (OA-2f)
Sample
  • Complex: Helicobacter pylori Vacuolating Cytotoxin A Oligomeric Assembly 2f (OA-2f)
Biological speciesHelicobacter pylori (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.5 Å
AuthorsZhang K / Zhang H / Li S / Au S / Chiu W
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, 2019-
Header (metadata) releaseMar 6, 2019-
Map releaseMar 27, 2019-
UpdateApr 17, 2019-
Current statusApr 17, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0548.png

Downloads & links

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Map

FileDownload / File: emd_0548.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHelicobacter pylori Vacuolating Cytotoxin A Oligomeric Assembly 2f (OA-2f)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 384 pix.
= 407.04 Å		1.06 Å/pix.
x 384 pix.
= 407.04 Å		1.06 Å/pix.
x 384 pix.
= 407.04 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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3 / Movie #1: 0.3
Minimum - Maximum-0.16100569 - 0.9376766
Average (Standard dev.)0.009521019 (±0.056656472)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 407.03998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z407.040407.040407.040
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.1610.9380.010

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Supplemental data

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Sample components

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Entire : Helicobacter pylori Vacuolating Cytotoxin A Oligomeric Assembly 2...

EntireName: Helicobacter pylori Vacuolating Cytotoxin A Oligomeric Assembly 2f (OA-2f)
Components
  • Complex: Helicobacter pylori Vacuolating Cytotoxin A Oligomeric Assembly 2f (OA-2f)

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Supramolecule #1: Helicobacter pylori Vacuolating Cytotoxin A Oligomeric Assembly 2...

SupramoleculeName: Helicobacter pylori Vacuolating Cytotoxin A Oligomeric Assembly 2f (OA-2f)
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Helicobacter pylori (bacteria) / Strain: 60190
Molecular weightExperimental: 88 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 7.4
GridSupport film - Material: CARBON / Support film - topology: CONTINUOUS / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
DetailsVacA OA-2f

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-30 / Number grids imaged: 3 / Number real images: 13708 / Average exposure time: 6.0 sec. / Average electron dose: 7.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 540214
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Final reconstructionApplied symmetry - Point group: D6 (2x6 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 15043
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final 3D classificationNumber classes: 10 / Software - Name: RELION (ver. 2)

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