[English] 日本語
Yorodumi
- PDB-4v3m: Membrane bound pleurotolysin prepore (TMH2 helix lock) trapped wi... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 4v3m
TitleMembrane bound pleurotolysin prepore (TMH2 helix lock) trapped with engineered disulphide cross-link
Components
  • PLEUROTOLYSIN A
  • PLEUROTOLYSIN B
KeywordsTRANSPORT PROTEIN / MACPF/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 / Hemolysin, aegerolysin type / Aegerolysin / Membrane attack complex/perforin (MACPF) domain profile. / MAC/Perforin domain / Membrane attack complex component/perforin (MACPF) domain
Similarity search - Domain/homology
Pleurotolysin B / Pleurotolysin A
Similarity search - Component
Biological speciesPLEUROTUS OSTREATUS (oyster mushroom)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 17 Å
Model type detailsCA ATOMS ONLY, CHAIN A, B, C
AuthorsLukoyanova, N. / Kondos, S.C. / Farabella, I. / Law, R.H.P. / Reboul, C.F. / Caradoc-Davies, T.T. / Spicer, B.A. / Kleifeld, O. / Perugini, M. / Ekkel, S. ...Lukoyanova, N. / Kondos, S.C. / Farabella, I. / Law, R.H.P. / Reboul, C.F. / Caradoc-Davies, T.T. / Spicer, B.A. / Kleifeld, O. / Perugini, M. / Ekkel, S. / Hatfaludi, T. / Oliver, K. / Hotze, E.M. / Tweten, R.K. / Whisstock, J.C. / Topf, M. / Dunstone, M.A. / Saibil, H.R.
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 20, 2014Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 18, 2015Provider: repository / Type: Initial release
Revision 1.1Aug 23, 2017Group: Data collection / Refinement description / Category: em_3d_fitting / em_software
Item: _em_3d_fitting.target_criteria / _em_software.fitting_id ..._em_3d_fitting.target_criteria / _em_software.fitting_id / _em_software.image_processing_id / _em_software.name

-
Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
  • Download
  • Simplified surface model + fitted atomic model
  • EMDB-2795
  • Imaged by Jmol
  • Download
  • Superimposition on EM map
  • EMDB-2795
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: PLEUROTOLYSIN A
B: PLEUROTOLYSIN A
C: PLEUROTOLYSIN B


Theoretical massNumber of molelcules
Total (without water)86,8663
Polymers86,8663
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Number of models20

-
Components

#1: Protein PLEUROTOLYSIN A / Coordinate model: Cα atoms only


Mass: 14852.545 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) PLEUROTUS OSTREATUS (oyster mushroom) / Plasmid: PET3A / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / Variant (production host): CODON PLUS PLYSS (NOVAGEN) / References: UniProt: Q8X1M9
#2: Protein PLEUROTOLYSIN B / Coordinate model: Cα atoms only


Mass: 57160.996 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) PLEUROTUS OSTREATUS (oyster mushroom) / Plasmid: PUC57, PET3A / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / Variant (production host): CODON PLUS PLYSS (NOVAGEN) / References: UniProt: Q5W9E8

-
Experimental details

-
Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

-
Sample preparation

ComponentName: PLEUROTOLYSIN PREPORE ON LIPOSOMES (TMH2 HELIX LOCK) TRAPPED WITH ENGINEERED DISULPHIDE
Type: COMPLEX
Buffer solutionName: 50 MM NACL, 20 MM HEPES / pH: 7.4 / Details: 50 MM NACL, 20 MM HEPES
SpecimenConc.: 0.02 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE
Details: VITRIFICATION 1 -- CRYOGEN- ETHANE, HUMIDITY- 80,INSTRUMENT- FEI VITROBOT MARK III, METHOD- PLEUROTOLYSIN A WAS FIRST ADDED TO SPHINGOMYELIN-CHOLESTEROL LIPOSOMES AT A MOLAR RATIO OF 1 TO ...Details: VITRIFICATION 1 -- CRYOGEN- ETHANE, HUMIDITY- 80,INSTRUMENT- FEI VITROBOT MARK III, METHOD- PLEUROTOLYSIN A WAS FIRST ADDED TO SPHINGOMYELIN-CHOLESTEROL LIPOSOMES AT A MOLAR RATIO OF 1 TO 2000 ROTEIN 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 TO 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 PERCENT HUMIDITY.

-
Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300 / Date: Jan 30, 2013
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Calibrated magnification: 76148 X / Nominal defocus max: 3700 nm / Nominal defocus min: 700 nm / Cs: 2.3 mm
Specimen holderTemperature: 94 K
Image recordingElectron dose: 25 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)
Image scansNum. digital images: 154
Radiation wavelengthRelative weight: 1

-
Processing

EM software
IDNameCategory
1Flex-EMmodel fitting
2MODELLERmodel fitting
3TEMPymodel fitting
4UCSF Chimeramodel fitting
5IMAGIC3D reconstruction
6SPIDER3D reconstruction
CTF correctionDetails: ESTIMATED WITH CTFFIND3, THEN PHASES FLIPPED FOR EACH PARTICLE
SymmetryPoint symmetry: C13 (13 fold cyclic)
3D reconstructionMethod: ANGULAR RECONSTITUTION AND PROJECTION MATCHING / Resolution: 17 Å / Num. of particles: 722 / Nominal pixel size: 1.94 Å / Actual pixel size: 2 Å
Details: UBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2795. (DEPOSITION ID: 12858).
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient / Details: METHOD--RIGID BODY
Atomic model buildingPDB-ID: 4OEB
RefinementHighest resolution: 17 Å
Refinement stepCycle: LAST / Highest resolution: 17 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms658 0 0 0 658

+
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