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- PDB-6rb9: The pore structure of Clostridium perfringens epsilon toxin -

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Basic information

Entry
Database: PDB / ID: 6rb9
TitleThe pore structure of Clostridium perfringens epsilon toxin
ComponentsEpsilon-toxin type B
KeywordsTOXIN / enterotoxaemia / beta-PFT / epsilon toxin / CryoEM
Function / homologyEpsilon toxin / Aerolysin-like toxin / Clostridium epsilon toxin ETX/Bacillus mosquitocidal toxin MTX2 / : / toxin activity / Epsilon-toxin type B
Function and homology information
Biological speciesClostridium perfringens B (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsSavva, C.G. / Clark, A.R. / Naylor, C.E. / Popoff, M.R. / Moss, D.S. / Basak, A.K. / Titball, R.W. / Bokori-Brown, M.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome TrustWT089618MA United Kingdom
Medical Research Council (United Kingdom)MC-A021-53019 United Kingdom
CitationJournal: Nat Commun / Year: 2019
Title: The pore structure of Clostridium perfringens epsilon toxin.
Authors: Christos G Savva / Alice R Clark / Claire E Naylor / Michel R Popoff / David S Moss / Ajit K Basak / Richard W Titball / Monika Bokori-Brown /
Abstract: Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens, is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in ...Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens, is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in multiple sclerosis in humans. Etx is a member of the aerolysin family of β-PFTs (aβ-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double β-barrel, a common feature of the aβ-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications.
History
DepositionApr 9, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 19, 2019Provider: repository / Type: Initial release
Revision 1.1Jun 26, 2019Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / database_PDB_rev / database_PDB_rev_record / em_admin / pdbx_database_proc
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 / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.2Dec 18, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
Revision 1.3Jul 29, 2020Group: Data collection / Category: em_imaging_optics
Item: _em_imaging_optics.chr_aberration_corrector / _em_imaging_optics.phase_plate

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Structure visualization

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Assembly

Deposited unit
A: Epsilon-toxin type B
B: Epsilon-toxin type B
C: Epsilon-toxin type B
D: Epsilon-toxin type B
E: Epsilon-toxin type B
F: Epsilon-toxin type B
G: Epsilon-toxin type B


Theoretical massNumber of molelcules
Total (without water)225,6437
Polymers225,6437
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area39260 Å2
ΔGint-161 kcal/mol
Surface area75230 Å2
MethodPISA

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Components

#1: Protein
Epsilon-toxin type B


Mass: 32234.695 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium perfringens B (bacteria) / Gene: etxB / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta 2 / References: UniProt: Q02307

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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: Epsilon toxin / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.2254 MDa / Experimental value: NO
Source (natural)Organism: Clostridium perfringens B (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: Rosetta 2 / Plasmid: pHis-Parallel1
Buffer solutionpH: 7.1
Details: DPBS containing 20 mM imidazole and 0.02% (w/v) DDM
SpecimenConc.: 0.013 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 40 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: AutoCTF
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 X / Nominal defocus max: 800 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 60 sec. / Electron dose: 32 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 835
EM imaging opticsChromatic aberration corrector: Volta phase plate was used. Just prior to data collection, the phase plate was inserted and the un-scattered beam was made parallel by observing the Ronchigram in the ...Chromatic aberration corrector: Volta phase plate was used. Just prior to data collection, the phase plate was inserted and the un-scattered beam was made parallel by observing the Ronchigram in the back focal plane of the objective
Phase plate: VOLTA PHASE PLATE
Image scansWidth: 4096 / Height: 4096

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3particle selection
2EPUimage acquisition
4GctfCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 263672
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 25525 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: A model was built into the 3.2 A map by initially docking the receptor-binding domain of the wild type Etx crystal structure (PDB: 1UYJ) and then extending this, building ab initio using Coot.

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