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- EMDB-33215: Cryo-EM reconstruction of complete transmembrane channel E289A mu... -

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Entry
Database: EMDB / ID: EMD-33215
TitleCryo-EM reconstruction of complete transmembrane channel E289A mutant Vibrio cholerae Cytolysin
Map dataCryo-EM reconstruction of complete transmembrane channel E289A mutant Vibrio cholerae Cytolysin
Sample
  • Complex: Cryo-EM reconstruction of complete transmembrane channel E289A mutant Vibrio cholerae Cytolysin
KeywordsVibrio cholerae Cytolysin (VCC) / Pore-forming toxin (PFT) / transmembrane channel / membrane / cryo-EM / TOXIN
Function / homology
Function and homology information


cytolysis in another organism / extracellular region
Similarity search - Function
Hemolytic toxin, N-terminal / Hemolytic toxin, N-terminal domain superfamily / Hemolysin, pre-stem domain / Hemolytic toxin N terminal / Hemolysin, beta-prism lectin / Beta-prism lectin / Leukocidin/Hemolysin toxin / Leukocidin/Hemolysin toxin family / Leukocidin/porin MspA superfamily / Jacalin-like lectin domain superfamily ...Hemolytic toxin, N-terminal / Hemolytic toxin, N-terminal domain superfamily / Hemolysin, pre-stem domain / Hemolytic toxin N terminal / Hemolysin, beta-prism lectin / Beta-prism lectin / Leukocidin/Hemolysin toxin / Leukocidin/Hemolysin toxin family / Leukocidin/porin MspA superfamily / Jacalin-like lectin domain superfamily / Ricin-type beta-trefoil / Lectin domain of ricin B chain profile. / Ricin B, lectin domain / Ricin B-like lectins
Similarity search - Domain/homology
Biological speciesVibrio cholerae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsMondal AK / Sengupta N / Singh M / Lata K / Lahiri I / Dutta S / Chattopadhyay K
Funding support India, 2 items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)BT/INF/22/SP22844/2017 India
Department of Science & Technology (DST, India)SR/FST/LSII-039/2015 India
CitationJournal: J Biol Chem / Year: 2022
Title: Glu289 residue in the pore-forming motif of Vibrio cholerae cytolysin is important for efficient β-barrel pore formation.
Authors: Anish Kumar Mondal / Nayanika Sengupta / Mahendra Singh / Rupam Biswas / Kusum Lata / Indrajit Lahiri / Somnath Dutta / Kausik Chattopadhyay /
Abstract: Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging β-barrel pore-forming toxin. Upon binding to the target membranes, VCC monomers first assemble into oligomeric prepore intermediates and ...Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging β-barrel pore-forming toxin. Upon binding to the target membranes, VCC monomers first assemble into oligomeric prepore intermediates and subsequently transform into transmembrane β-barrel pores. VCC harbors a designated pore-forming motif, which, during oligomeric pore formation, inserts into the membrane and generates a transmembrane β-barrel scaffold. It remains an enigma how the molecular architecture of the pore-forming motif regulates the VCC pore-formation mechanism. Here, we show that a specific pore-forming motif residue, E289, plays crucial regulatory roles in the pore-formation mechanism of VCC. We find that the mutation of E289A drastically compromises pore-forming activity, without affecting the structural integrity and membrane-binding potential of the toxin monomers. Although our single-particle cryo-EM analysis reveals WT-like oligomeric β-barrel pore formation by E289A-VCC in the membrane, we demonstrate that the mutant shows severely delayed kinetics in terms of pore-forming ability that can be rescued with elevated temperature conditions. We find that the pore-formation efficacy of E289A-VCC appears to be more profoundly dependent on temperature than that of the WT toxin. Our results suggest that the E289A mutation traps membrane-bound toxin molecules in the prepore-like intermediate state that is hindered from converting into the functional β-barrel pores by a large energy barrier, thus highlighting the importance of this residue for the pore-formation mechanism of VCC.
History
DepositionApr 14, 2022-
Header (metadata) releaseMay 4, 2022-
Map releaseMay 4, 2022-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: PDBj / Status: Released

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Structure visualization

Downloads & links

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Map

FileDownload / File: emd_33215.map.gz / Format: CCP4 / Size: 47.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of complete transmembrane channel E289A mutant Vibrio cholerae Cytolysin
Voxel sizeX=Y=Z: 0.92 Å
Density
Contour LevelBy AUTHOR: 0.114
Minimum - Maximum-0.28281015 - 0.5176444
Average (Standard dev.)0.0029716818 (±0.03685908)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions232232232
Spacing232232232
CellA=B=C: 213.44 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Cryo-EM reconstruction of complete transmembrane channel E289A mu...

EntireName: Cryo-EM reconstruction of complete transmembrane channel E289A mutant Vibrio cholerae Cytolysin
Components
  • Complex: Cryo-EM reconstruction of complete transmembrane channel E289A mutant Vibrio cholerae Cytolysin

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Supramolecule #1: Cryo-EM reconstruction of complete transmembrane channel E289A mu...

SupramoleculeName: Cryo-EM reconstruction of complete transmembrane channel E289A mutant Vibrio cholerae Cytolysin
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Vibrio cholerae (bacteria)

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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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.75 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 42124
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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