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- EMDB-2698: The cryoEM structure of Monalysin Toxin -

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Basic information

Entry
Database: EMDB / ID: EMD-2698
TitleThe cryoEM structure of Monalysin Toxin
Map dataReconstruction of Monalysin
Sample
  • Sample: Monalysin
  • Protein or peptide: Monalysin
KeywordsMonalysin / cryo electron microscopy / single-particle
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 17.0 Å
AuthorsLeone P / Bebeacua C / Opota O / Kellenberger C / Klaholz B / Cambillau C / Lemaitre B / Roussel A
CitationJournal: J Biol Chem / Year: 2015
Title: X-ray and Cryo-electron Microscopy Structures of Monalysin Pore-forming Toxin Reveal Multimerization of the Pro-form.
Authors: Philippe Leone / Cecilia Bebeacua / Onya Opota / Christine Kellenberger / Bruno Klaholz / Igor Orlov / Christian Cambillau / Bruno Lemaitre / Alain Roussel /
Abstract: β-Barrel pore-forming toxins (β-PFT), a large family of bacterial toxins, are generally secreted as water-soluble monomers and can form oligomeric pores in membranes following proteolytic cleavage ...β-Barrel pore-forming toxins (β-PFT), a large family of bacterial toxins, are generally secreted as water-soluble monomers and can form oligomeric pores in membranes following proteolytic cleavage and interaction with cell surface receptors. Monalysin has been recently identified as a β-PFT that contributes to the virulence of Pseudomonas entomophila against Drosophila. It is secreted as a pro-protein that becomes active upon cleavage. Here we report the crystal and cryo-electron microscopy structure of the pro-form of Monalysin as well as the crystal structures of the cleaved form and of an inactive mutant lacking the membrane-spanning region. The overall structure of Monalysin displays an elongated shape, which resembles those of β-pore-forming toxins, such as Aerolysin, but is devoid of a receptor-binding domain. X-ray crystallography, cryo-electron microscopy, and light-scattering studies show that pro-Monalysin forms a stable doughnut-like 18-mer complex composed of two disk-shaped nonamers held together by N-terminal swapping of the pro-peptides. This observation is in contrast with the monomeric pro-form of the other β-PFTs that are receptor-dependent for membrane interaction. The membrane-spanning region of pro-Monalysin is fully buried in the center of the doughnut, suggesting that upon cleavage of pro-peptides, the two disk-shaped nonamers can, and have to, dissociate to leave the transmembrane segments free to deploy and lead to pore formation. In contrast with other toxins, the delivery of 18 subunits at once, nearby the cell surface, may be used to bypass the requirement of receptor-dependent concentration to reach the threshold for oligomerization into the pore-forming complex.
History
DepositionJul 1, 2014-
Header (metadata) releaseDec 24, 2014-
Map releaseDec 24, 2014-
UpdateDec 24, 2014-
Current statusDec 24, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 15
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 15
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2698.map.gz / Format: CCP4 / Size: 11.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Monalysin
Voxel sizeX=Y=Z: 1.92 Å
Density
Contour LevelBy AUTHOR: 15.0 / Movie #1: 15
Minimum - Maximum0.0 - 26.511838910000002
Average (Standard dev.)0.86904889 (±4.25143194)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions144144144
Spacing144144144
CellA=B=C: 276.47998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.921.921.92
M x/y/z144144144
origin x/y/z0.0000.0000.000
length x/y/z276.480276.480276.480
α/β/γ90.00090.00090.000
start NX/NY/NZ-180-180-179
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS144144144
D min/max/mean-0.00026.5120.869

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Supplemental data

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Sample components

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Entire : Monalysin

EntireName: Monalysin
Components
  • Sample: Monalysin
  • Protein or peptide: Monalysin

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Supramolecule #1000: Monalysin

SupramoleculeName: Monalysin / type: sample / ID: 1000 / Oligomeric state: 18mer / Number unique components: 1

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Macromolecule #1: Monalysin

MacromoleculeName: Monalysin / type: protein_or_peptide / ID: 1 / Number of copies: 9 / Oligomeric state: 9mer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

Concentration0.5 mg/mL
BufferpH: 8 / Details: 10mM Tris, 500mM NaCl
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK I / Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 0.002 µm / Nominal defocus min: 0.001 µm / Nominal magnification: 60000
Sample stageSpecimen holder model: PHILIPS ROTATION HOLDER
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification.
DateSep 1, 2012
Image recordingDigitization - Sampling interval: 1.92 µm / Number real images: 220 / Average electron dose: 10 e/Å2
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Images
Final reconstructionApplied symmetry - Point group: D9 (2x9 fold dihedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: OTHER / Software - Name: Eman2, Spider, Xmipp / Number images used: 12000

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