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- PDB-8jc7: Cryo-EM structure of Vibrio campbellii alpha-hemolysin -

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Basic information

Entry
Database: PDB / ID: 8jc7
TitleCryo-EM structure of Vibrio campbellii alpha-hemolysin
ComponentsHemolysin
KeywordsTOXIN / Vibrio campbellii alpha-hemolysin / Pore-forming toxins (PFTs) / Calcium-dependent oligomerization / Membrane association / Structure-function relationship
Function / homology: / :
Function and homology information
Biological speciesVibrio campbellii (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.06 Å
AuthorsWang, C.H. / Yeh, M.K. / Ho, M.C. / Lin, S.M.
Funding support Taiwan, 2items
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, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 27, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 4, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Hemolysin
B: Hemolysin
C: Hemolysin
D: Hemolysin
E: Hemolysin
F: Hemolysin
G: Hemolysin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)565,05921
Polymers564,5057
Non-polymers55414
Water10,647591
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Hemolysin


Mass: 80643.578 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio campbellii (bacteria) / Gene: DSB67_23490 / Production host: Escherichia coli (E. coli) / Strain (production host): Rossetta-gami 2 / References: UniProt: A0A344KRS4
#2: Chemical
ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 591 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: heptameric ring-shaped complex of Vibrio campbellii alpha-hemolysin
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Vibrio campbellii (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: Rossetta-gami 2
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELD / Nominal magnification: 81000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: EPU / Version: 2.7.0 / Category: image acquisition
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 2.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 142403 / Symmetry type: POINT

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