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- PDB-4v3a: Membrane bound pleurotolysin prepore (TMH1 lock) trapped with eng... -

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Basic information

Entry
Database: PDB / ID: 4v3a
TitleMembrane bound pleurotolysin prepore (TMH1 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 / Fungal MACPF-like domain / Hemolysin, aegerolysin type / Aegerolysin / Membrane attack complex/perforin (MACPF) domain profile. / 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: 15 Å
Model type detailsCA ATOMS ONLY, CHAIN A, B, C
AuthorsLukoyanova, N. / Kondos, S.C. / Farabella, I. / Law, R.H.P. / Reboul, C.F. / CaradocDavies, 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. / CaradocDavies, 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 17, 2014Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 18, 2015Provider: repository / Type: Initial release
Revision 1.1Aug 30, 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
Revision 1.2May 8, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-2794
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  • Superimposition on EM map
  • EMDB-2794
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Structure viewerMolecule:
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Assembly

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


Theoretical massNumber of molelcules
Total (without water)72,6163
Polymers72,6163
Non-polymers00
Water00
1


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

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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: 42910.660 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

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: PLEUROTOLYSIN PREPORE ON LIPOSOMES (TMH1 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 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 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.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300 / Date: Jul 1, 2011
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 59000 X / Calibrated magnification: 76148 X / Nominal defocus max: 3600 nm / Nominal defocus min: 900 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: 274
Radiation wavelengthRelative weight: 1

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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: 15 Å / Num. of particles: 1150 / Nominal pixel size: 1.94 Å / Actual pixel size: 2 Å
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2794. (DEPOSITION ID: 12853).
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

4oeb


Accession code: 4OEB / Source name: PDB / Type: experimental model
RefinementHighest resolution: 15 Å
Refinement stepCycle: LAST / Highest resolution: 15 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms658 0 0 0 658

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