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- EMDB-36150: Cryo-EM structure of Vibrio campbellii alpha-hemolysin -

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Basic information

Entry
Database: EMDB / ID: EMD-36150
TitleCryo-EM structure of Vibrio campbellii alpha-hemolysin
Map data
Sample
  • Complex: heptameric ring-shaped complex of Vibrio campbellii alpha-hemolysin
    • Protein or peptide: Hemolysin
  • Ligand: POTASSIUM IONPotassium
  • Ligand: CALCIUM IONCalcium
  • Ligand: water
KeywordsVibrio campbellii alpha-hemolysin / Pore-forming toxins (PFTs) / Calcium-dependent oligomerization / Membrane association / Structure-function relationship / TOXIN
Function / homology:
Function and homology information
Biological speciesVibrio campbellii (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.06 Å
AuthorsWang CH / Yeh MK / Ho MC / Lin SM
Funding support Taiwan, 2 items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, Taiwan) Taiwan
Academia Sinica (Taiwan) Taiwan
CitationJournal: Nat Commun / Year: 2023
Title: Structural basis for calcium-stimulating pore formation of Vibrio α-hemolysin.
Authors: Yu-Chuan Chiu / Min-Chi Yeh / Chun-Hsiung Wang / Yu-An Chen / Hsiang Chang / Han-You Lin / Meng-Chiao Ho / Shih-Ming Lin /
Abstract: Vibrio α-hemolysins (αHLs) are β-pore-forming toxins secreted by Vibrio pathogens, crucial for the facilitation of bacterial infections through host cell lysis. These toxins are produced as ...Vibrio α-hemolysins (αHLs) are β-pore-forming toxins secreted by Vibrio pathogens, crucial for the facilitation of bacterial infections through host cell lysis. These toxins are produced as inactive precursors, requiring proteolytic maturation and membrane association for activation within host tissues. Here, we investigate Vibrio campbellii αHL (VcαHL), and establish that its hemolytic activity is significantly stimulated by calcium ions, with an EC that aligns with physiological calcium concentrations. Furthermore, we illustrate the vital contribution of calcium ions to the oligomerization of VcαHL on membranes. Using X-ray crystallography and cryo-electron microscopy, we decipher both the immature and assembled structures of VcαHL and elucidate the conformational changes corresponding to toxin assembly. We also identify a calcium-binding module that is integral for VcαHL's calcium-dependent activation. These findings provide insights into the regulatory mechanisms of VcαHL and have the potential to inform the development of targeted therapeutic strategies against Vibrio infections.
History
DepositionMay 10, 2023-
Header (metadata) releaseSep 27, 2023-
Map releaseSep 27, 2023-
UpdateOct 4, 2023-
Current statusOct 4, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36150.png

Downloads & links

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Map

FileDownload / File: emd_36150.map.gz / Format: CCP4 / Size: 1000 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.5305 Å
Density
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-2.0220716 - 3.04348
Average (Standard dev.)0.0012857033 (±0.084142916)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions640640640
Spacing640640640
CellA=B=C: 339.52 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: unsharpen map

Fileemd_36150_additional_1.map
Annotationunsharpen map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_36150_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_36150_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : heptameric ring-shaped complex of Vibrio campbellii alpha-hemolysin

EntireName: heptameric ring-shaped complex of Vibrio campbellii alpha-hemolysin
Components
  • Complex: heptameric ring-shaped complex of Vibrio campbellii alpha-hemolysin
    • Protein or peptide: Hemolysin
  • Ligand: POTASSIUM IONPotassium
  • Ligand: CALCIUM IONCalcium
  • Ligand: water

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Supramolecule #1: heptameric ring-shaped complex of Vibrio campbellii alpha-hemolysin

SupramoleculeName: heptameric ring-shaped complex of Vibrio campbellii alpha-hemolysin
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Vibrio campbellii (bacteria)

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Macromolecule #1: Hemolysin

MacromoleculeName: Hemolysin / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Vibrio campbellii (bacteria)
Molecular weightTheoretical: 80.643578 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNINEPSGEA ANIISQAADS HAMKYYNAAD WQAEDNALPS LAELRDLVIN QQKSVLFDFS QNSDADGQAE MQAQFRKTYG VGFANQFIF ITEHKGELLF TPFEHSEEVD PKLLEAPLTT RSGLKSTAPT NSETSTLPHV AFYISVNRPI SDEECTFDNS W LWKDEKGS ...String:
MNINEPSGEA ANIISQAADS HAMKYYNAAD WQAEDNALPS LAELRDLVIN QQKSVLFDFS QNSDADGQAE MQAQFRKTYG VGFANQFIF ITEHKGELLF TPFEHSEEVD PKLLEAPLTT RSGLKSTAPT NSETSTLPHV AFYISVNRPI SDEECTFDNS W LWKDEKGS RPFCKDANIS LIYRVNLERS LQYGIVGSAT PNAKIVRISL DDDSSGAGIH LNDQLSYRRF GASYTTLDAY FR EWSTDAI AQDYRFVFKT SNNKAEILET FPIDNLNVKY EKRKQSGFEL GVTGGAEVSE DGPKAKLEAR ASITQSRWLT YNT QDYRVE RNAKNAQTVS FTWNRQEYAT AESLLNRSTD ALWVDTYPVD VNRISPLSYA SFVPKMDVIY KASDTETGST DFII DSSVN IRPIYNGAYK HYYVVGAHQS YHGFENSPRR RITKSASFTV DWDHPVFTGG RPVNLQLASF NNRCVQVDAQ SRLTA NTCD DQQSAQSFIY DQLGRYVSAS NTELCLDGAA LDVLQTCNQN LTQRWEWRKN TDELTNVYSG ESLGHDKQTG ELGLYA SSN DAVSLRTITA YTNVFNVQKS SPILGYTQGK MNQQSVGQNY RLYVREGSAI DALGTASDLL VGGNGGSLTS VDLSGVK SI TATSGDFQYG GQQLVALTFT YQDGRQQMVG SKAHVTNAHE DRFDLPDAAK ITQLNIWADD WLVKGVQFDL NLEHHHHH H

UniProtKB: UNIPROTKB: A0A344KRS4

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Macromolecule #2: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 2 / Number of copies: 7 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Macromolecule #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 7 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 591 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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: 7.6
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.06 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 142403
FSC plot (resolution estimation)

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