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- EMDB-43271: Cryo-EM structure of the Helicobacter pylori VacA hexamer that wa... -

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Basic information

Entry
Database: EMDB / ID: EMD-43271
TitleCryo-EM structure of the Helicobacter pylori VacA hexamer that was detergent solubilized from membrane, C6 symmetry applied
Map dataVacA hexamer solubilized from LUVs with DDM. C6 symmetry applied.
Sample
  • Complex: Helicobacter pylori VacA hexamer detergent-solubilized from liposomes, C6 symmetry applied
Keywordspore-forming toxin / membrane-associated protein / TOXIN
Biological speciesHelicobacter pylori (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.7 Å
AuthorsConnolly SM / Ohi MD
Funding support United States, 9 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI039657 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI118932 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA116087 United States
National Institutes of Health/Office of the DirectorS10OD030275 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM08320 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM007315 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F31GM139291 United States
American Heart Association905705 United States
Department of Veterans Affairs (VA, United States)5I01BX004447 United States
CitationJournal: J Mol Biol / Year: 2024
Title: Structural Analysis of Membrane-associated Forms of Helicobacter pylori VacA Toxin.
Authors: Sarah M Connolly / Amanda L Erwin / Megan Sabb / Jessica L Hanks / Louise Chang / Rachel M Torrez / Georgia C Caso / Anne M Campbell / Shyamal Mosalaganti / Timothy L Cover / Melanie D Ohi /
Abstract: Helicobacter pylori colonizes the stomach in about half of the human population, leading to an increased risk of peptic ulcer disease and gastric cancer. H. pylori secretes an 88 kDa VacA toxin that ...Helicobacter pylori colonizes the stomach in about half of the human population, leading to an increased risk of peptic ulcer disease and gastric cancer. H. pylori secretes an 88 kDa VacA toxin that contributes to pathogenesis. VacA assembles into oligomeric complexes in solution and forms anion-selective channels in cell membranes. Cryo-electron microscopy (cryo-EM) analyses of VacA oligomers in solution provided insights into VacA oligomerization but failed to reveal the structure of the hydrophobic N-terminal region predicted to be a pore-forming domain. In this study, we incubated VacA with liposomes and used single particle cryo-EM to analyze detergent-extracted VacA oligomers. A 3D structure of detergent-solubilized VacA hexamers revealed the presence of six α-helices extending from the center of the oligomers, a feature not observed in previous studies of water-soluble VacA oligomers. Cryo-electron tomography analysis and 2D averages of VacA associated with liposomes confirmed that central regions of the membrane-associated VacA oligomers can insert into the lipid bilayer. However, insertion is heterogenous, with some membrane-associated oligomers appearing only partially inserted and others sitting on top of the bilayer. These studies indicate that VacA undergoes a conformational change when contacting the membrane and reveal an α-helical region positioned to extend into the membrane. Although the reported VacA 3D structure does not represent a selective anion channel, our combined single particle 3D analysis, cryo-electron tomography, and modeling allow us to propose a model for the structural organization of the VacA N-terminus in the context of a hexamer as it inserts into the membrane.
History
DepositionJan 5, 2024-
Header (metadata) releaseJan 24, 2024-
Map releaseJan 24, 2024-
UpdateJan 24, 2024-
Current statusJan 24, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43271.png

Downloads & links

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Map

FileDownload / File: emd_43271.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationVacA hexamer solubilized from LUVs with DDM. C6 symmetry applied.
Voxel sizeX=Y=Z: 2.007 Å
Density
Contour LevelBy AUTHOR: 0.198
Minimum - Maximum-0.50483245 - 1.148653
Average (Standard dev.)0.00023063425 (±0.026028067)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 602.1 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: VacA hexamer solubilized from LUVs with DDM. C6...

Fileemd_43271_half_map_1.map
AnnotationVacA hexamer solubilized from LUVs with DDM. C6 symmetry applied. Map A.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: VacA hexamer solubilized from LUVs with DDM. C6...

Fileemd_43271_half_map_2.map
AnnotationVacA hexamer solubilized from LUVs with DDM. C6 symmetry applied. Map B.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Helicobacter pylori VacA hexamer detergent-solubilized from lipos...

EntireName: Helicobacter pylori VacA hexamer detergent-solubilized from liposomes, C6 symmetry applied
Components
  • Complex: Helicobacter pylori VacA hexamer detergent-solubilized from liposomes, C6 symmetry applied

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Supramolecule #1: Helicobacter pylori VacA hexamer detergent-solubilized from lipos...

SupramoleculeName: Helicobacter pylori VacA hexamer detergent-solubilized from liposomes, C6 symmetry applied
type: complex / ID: 1 / Parent: 0
Details: VacA hexamer bound to large unilamellar vesicles (LUVs) and solubilized by DDM
Source (natural)Organism: Helicobacter pylori (bacteria) / Strain: 60190

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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

BufferpH: 7.3
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 51.61 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.0 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Ab initio model
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 6.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 20539
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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