[English] 日本語
Yorodumi
- EMDB-2796: Membrane bound pleurotolysin prepore (TMH2 strand lock) trapped w... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-2796
TitleMembrane bound pleurotolysin prepore (TMH2 strand lock) trapped with engineered disulphide cross-link
Map dataThis TMH2 strand disulphide trap mutant (V277C, K291C) was engineered on a background PlyB variant that lacks the wildtype cysteine (C487A) in order to avoid incorrect disulphide bond formation.
Sample
  • Sample: pleurotolysin prepore on liposomes (TMH2 strand lock) trapped with engineered disulphide
  • 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: 14.0 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 19, 2015-
Current statusAug 19, 2015Processing site: PDBe / Status: Released

-
Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0002
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0002
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-4v3n
  • Surface level: 0.0002
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4v3n
  • Imaged by Jmol
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2796_ima...

Downloads & links

-
Map

FileDownload / File: emd_2796.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis TMH2 strand disulphide trap mutant (V277C, K291C) was engineered on a background PlyB variant that lacks the wildtype cysteine (C487A) in order to avoid incorrect disulphide bond formation.
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: 0.0002 / Movie #1: 0.0002
Minimum - Maximum-0.00209311 - 0.00225684
Average (Standard dev.)0.00001894 (±0.00021508)
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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0020.0020.000

-
Supplemental data

-
Sample components

-
Entire : pleurotolysin prepore on liposomes (TMH2 strand lock) trapped wit...

EntireName: pleurotolysin prepore on liposomes (TMH2 strand lock) trapped with engineered disulphide
Components
  • Sample: pleurotolysin prepore on liposomes (TMH2 strand lock) trapped with engineered disulphide
  • Protein or peptide: pleurotolysin A
  • Protein or peptide: pleurotolysin B

-
Supramolecule #1000: pleurotolysin prepore on liposomes (TMH2 strand lock) trapped wit...

SupramoleculeName: pleurotolysin prepore on liposomes (TMH2 strand lock) trapped with engineered disulphide
type: sample / ID: 1000
Details: 13 fold symmetrical ring oligomers of 26 PlyA and 13 PlyB (C487A, V277C, K291C variant) molecules on liposomes
Number unique components: 2

-
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

-
Macromolecule #2: pleurotolysin B

MacromoleculeName: pleurotolysin B / type: protein_or_peptide / ID: 2 / Name.synonym: PlyB / Details: C487A, V277C, K291C variant / 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

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

Concentration0.02 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.

-
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
DateMar 5, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 210 / 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: 3.4 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

-
Image processing

Details2,700 prepore side views with 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.
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: 14.0 Å / Resolution method: OTHER / Software - Name: Imagic, Spider / Details: SPIDER operation BP RP was used for reconstruction / Number images used: 1110

-
Atomic model buiding 1

Initial modelPDB ID:

4oeb

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation coefficient
Output model

PDB-4v3n:
Membrane bound pleurotolysin prepore (TMH2 strand lock) trapped with engineered disulphide cross-link

-
Atomic model buiding 2

Initial modelPDB ID:

4oej

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

PDB-4v3n:
Membrane bound pleurotolysin prepore (TMH2 strand lock) trapped with engineered disulphide cross-link

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more