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- EMDB-4789: The pore structure of Clostridium perfringens epsilon toxin -

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Basic information

Entry
Database: EMDB / ID: EMD-4789
TitleThe pore structure of Clostridium perfringens epsilon toxin
Map data
Sample
  • Complex: Epsilon toxin
    • Protein or peptide: Epsilon-toxin type B
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsSavva CG / Clark AR / Naylor CE / Popoff MR / Moss DS / Basak AK / Titball RW / Bokori-Brown M
Funding support United Kingdom, 2 items
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, 2019-
Header (metadata) releaseJun 19, 2019-
Map releaseJun 19, 2019-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6rb9
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4789.png

Downloads & links

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Map

FileDownload / File: emd_4789.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.13437322 - 0.24674426
Average (Standard dev.)0.00046999514 (±0.0075532333)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 235.40001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z235.400235.400235.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ858858858
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.1340.2470.000

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Supplemental data

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Half map: #1

Fileemd_4789_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_4789_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 : Epsilon toxin

EntireName: Epsilon toxin
Components
  • Complex: Epsilon toxin
    • Protein or peptide: Epsilon-toxin type B

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Supramolecule #1: Epsilon toxin

SupramoleculeName: Epsilon toxin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Clostridium perfringens B (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta 2 / Recombinant plasmid: pHis-Parallel1
Molecular weightTheoretical: 225.4 KDa

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Macromolecule #1: Epsilon-toxin type B

MacromoleculeName: Epsilon-toxin type B / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Clostridium perfringens B (bacteria)
Molecular weightTheoretical: 32.234695 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSYYHHHHHH DYDIPTTENL YFQGAMASYD NVDTLIEKGR YNTKYNYLKR MEKYYPNAMA YFDKVTINPQ GNDFYINNPK VELDGEPSM NYLEDVYVGK ALLTNDTQQE QKLKSQSFTC KNTDTVTATT THTVGTSIQA TAKFTVPFNE TGVSLTTSYS F ANTNTNTN ...String:
MSYYHHHHHH DYDIPTTENL YFQGAMASYD NVDTLIEKGR YNTKYNYLKR MEKYYPNAMA YFDKVTINPQ GNDFYINNPK VELDGEPSM NYLEDVYVGK ALLTNDTQQE QKLKSQSFTC KNTDTVTATT THTVGTSIQA TAKFTVPFNE TGVSLTTSYS F ANTNTNTN SKEITHNVPS QDILVPANTT VEVIAYLKKV NVKGNVKLVG QVSGSEWGEI PSYLAFPRDG YKFSLSDTVN KS DLNEDGT ININGKGNYS AVMGDELIVK VRNLNTNNVQ EYVIPVDKK

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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

Concentration0.013 mg/mL
BufferpH: 7.1
Details: DPBS containing 20 mM imidazole and 0.02% (w/v) DDM
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: 40 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 0.8 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 75000
Specialist opticsPhase plate: VOLTA PHASE PLATE
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 ...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
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsAutoCTF
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 835 / Average exposure time: 60.0 sec. / Average electron dose: 32.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 263672
CTF correctionSoftware - Name: Gctf
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7ahl


Details: Atomic model low-pass filtered and scaled to match EM data.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C7 (7 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 25525

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Atomic model buiding 1

DetailsA 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.
RefinementSpace: REAL / Protocol: OTHER
Output model

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

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