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- EMDB-2793: Membrane embedded pleurotolysin pore with 13 fold symmetry -

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Basic information

Entry
Database: EMDB / ID: EMD-2793
TitleMembrane embedded pleurotolysin pore with 13 fold symmetry
Map datamap was sharpened by reducing the amplitudes of low frequency components corresponding to spacings greater than 30 A and high frequency ones less than 10A
Sample
  • Sample: pleurotolysin pore in liposomes
  • Protein or peptide: pleurotolysin A
  • Protein or peptide: pleurotolysin B
KeywordsMACPF/CDC superfamily / pore-forming proteins
Function / homology
Function and homology information


hemolysis by symbiont of host erythrocytes
Similarity search - Function
Pleurotolysin B, C-terminal / Pleurotolysin B C-terminal domain / Fungal MACPF-like domain / Hemolysin, aegerolysin type / Hemolysin, aegerolysin type / Aegerolysin / Membrane attack complex/perforin (MACPF) domain profile. / Membrane attack complex component/perforin (MACPF) domain / Membrane attack complex component/perforin (MACPF) domain
Similarity search - Domain/homology
Pleurotolysin B / Pleurotolysin A
Similarity search - Component
Biological speciesPleurotus ostreatus (oyster mushroom)
Methodsingle particle reconstruction / cryo EM / Resolution: 11.0 Å
AuthorsLukoyanova N / Kondos SC / Farabella I / Law RHP / Reboul CF / Caradoc-Davies TT / Spicer BA / Kleifeld O / Perugini M / Ekkel S ...Lukoyanova N / Kondos SC / Farabella I / Law RHP / Reboul CF / Caradoc-Davies TT / Spicer BA / Kleifeld O / Perugini M / Ekkel S / Hatfaludi T / Oliver K / Hotze EM / Tweten RK / Whisstock JC / Topf M / Dunstone MA / Saibil HR
CitationJournal: PLoS Biol / Year: 2015
Title: Conformational changes during pore formation by the perforin-related protein pleurotolysin.
Authors: Natalya Lukoyanova / Stephanie C Kondos / Irene Farabella / Ruby H P Law / Cyril F Reboul / Tom T Caradoc-Davies / Bradley A Spicer / Oded Kleifeld / Daouda A K Traore / Susan M Ekkel / Ilia ...Authors: Natalya Lukoyanova / Stephanie C Kondos / Irene Farabella / Ruby H P Law / Cyril F Reboul / Tom T Caradoc-Davies / Bradley A Spicer / Oded Kleifeld / Daouda A K Traore / Susan M Ekkel / Ilia Voskoboinik / Joseph A Trapani / Tamas Hatfaludi / Katherine Oliver / Eileen M Hotze / Rodney K Tweten / James C Whisstock / Maya Topf / Helen R Saibil / Michelle A Dunstone /
Abstract: Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into ...Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ∼70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function.
History
DepositionOct 14, 2014-
Header (metadata) releaseNov 19, 2014-
Map releaseFeb 18, 2015-
UpdateAug 12, 2015-
Current statusAug 12, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4v2t
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4v2t
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2793_ima...

Downloads & links

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Map

FileDownload / File: emd_2793.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap was sharpened by reducing the amplitudes of low frequency components corresponding to spacings greater than 30 A and high frequency ones less than 10A
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2 Å/pix.
x 200 pix.
= 400. Å		2 Å/pix.
x 200 pix.
= 400. Å		2 Å/pix.
x 200 pix.
= 400. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25 / Movie #1: 0.25
Minimum - Maximum-3.04218602 - 4.09975386
Average (Standard dev.)0.00720952 (±0.31593281)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 400.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z222
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z400.000400.000400.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-3.0424.1000.007

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Supplemental data

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Sample components

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Entire : pleurotolysin pore in liposomes

EntireName: pleurotolysin pore in liposomes
Components
  • Sample: pleurotolysin pore in liposomes
  • Protein or peptide: pleurotolysin A
  • Protein or peptide: pleurotolysin B

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Supramolecule #1000: pleurotolysin pore in liposomes

SupramoleculeName: pleurotolysin pore in liposomes / type: sample / ID: 1000
Details: ring oligomers of 26 PlyA and 13 PlyB molecules on liposomes
Number unique components: 2

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Macromolecule #1: pleurotolysin A

MacromoleculeName: pleurotolysin A / type: protein_or_peptide / ID: 1 / Name.synonym: PlyA / Number of copies: 26 / Recombinant expression: Yes
Source (natural)Organism: Pleurotus ostreatus (oyster mushroom) / synonym: Oyster mushroom, White-rot fungus
Molecular weightTheoretical: 17 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Codon Plus pLysS (Novagen) / Recombinant plasmid: pET3a
SequenceUniProtKB: Pleurotolysin A / GO: hemolysis by symbiont of host erythrocytes / InterPro: Hemolysin, aegerolysin type

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Macromolecule #2: pleurotolysin B

MacromoleculeName: pleurotolysin B / type: protein_or_peptide / ID: 2 / Name.synonym: PlyB / Number of copies: 13 / Recombinant expression: Yes
Source (natural)Organism: Pleurotus ostreatus (oyster mushroom) / synonym: Oyster mushroom, White-rot fungus
Molecular weightTheoretical: 52 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Codon Plus pLysS (Novagen) / Recombinant plasmid: pUC57, pET3a
SequenceUniProtKB: Pleurotolysin B / GO: hemolysis by symbiont of host erythrocytes
InterPro: Membrane attack complex component/perforin (MACPF) domain

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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

Concentration0.01 mg/mL
BufferpH: 7.4 / Details: 50 mM NaCl, 20 mM Hepes
GridDetails: 300 mesh lacey copper grids
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Instrument: FEI VITROBOT MARK III
Method: Pleurotolysin A was first added to sphingomyelin/cholesterol liposomes at a molar ratio of 1:2000 protein to lipid in the above buffer. After 5 min incubation at room temperature, ...Method: Pleurotolysin A was first added to sphingomyelin/cholesterol liposomes at a molar ratio of 1:2000 protein to lipid in the above buffer. After 5 min incubation at room temperature, pleurotolysin B was added to the mixture at a molar ratio of 1:2 to pleurotolysin A. The mixture was incubated at 40 C or room temperature for 30 min after which 3.5 uL were placed on negatively glow discharged lacey grids and vitrified in liquid ethane using a Vitrobot. Blotting was carried out at 36 C and 80% humidity.

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 90 K / Max: 102 K / Average: 94 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 115,000 times magnification
DateJan 19, 2011
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 350 / Average electron dose: 25 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 76148 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.3 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 59000
Sample stageSpecimen holder: liquid nitrogen cooled / Specimen holder model: GATAN HELIUM
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Details2,500 pore top views and 14,500 pore side views with their adjacent membrane regions were extracted from liposome images and treated as single particles. These particles were initially used to separate 12- and 13-fold symmetries by angular reconstitution followed by competitive projection matching. 11,000 selected pore side views with estimated 13-fold symmetry were split randomly into 2 data sets, and again analysed independently.
CTF correctionDetails: Estimated with CTFFIND3, then phases flipped for each particle
Final reconstructionApplied symmetry - Point group: C13 (13 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.0 Å / Resolution method: OTHER / Software - Name: Imagic, Spider / Details: SPIDER operation BP RP was used for reconstruction / Number images used: 8700

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Atomic model buiding 1

Initial modelPDB ID:

4oeb

SoftwareName: Chimera, Flex-EM, Modeller
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-4v2t:
Membrane embedded pleurotolysin pore with 13 fold symmetry

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Atomic model buiding 2

Initial modelPDB ID:

4oej

SoftwareName: Chimera, Flex-EM, Modeller
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-4v2t:
Membrane embedded pleurotolysin pore with 13 fold symmetry

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