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- EMDB-2794: Membrane bound pleurotolysin prepore (TMH1 lock) trapped with eng... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-2794 | |||||||||
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Title | Membrane bound pleurotolysin prepore (TMH1 lock) trapped with engineered disulphide cross-link | |||||||||
![]() | This TMH1 disulphide trap mutant (F138C H221C) was engineered on a background PlyB variant that lacks the wildtype cysteine (C487A) in order to avoid incorrect disulphide bond formation. | |||||||||
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![]() | MACPF/CDC superfamily / pore-forming proteins | |||||||||
Function / homology | ![]() | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 15.0 Å | |||||||||
![]() | Lukoyanova 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 | |||||||||
![]() | ![]() 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 |
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Structure visualization
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 28.6 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 14.3 KB 14.3 KB | Display Display | ![]() |
Images | ![]() | 365.9 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 4v3aMC ![]() 2793C ![]() 2795C ![]() 2796C ![]() 4oebC ![]() 4oejC ![]() 4ov8C ![]() 4v2tC ![]() 4v3mC ![]() 4v3nC M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | This TMH1 disulphide trap mutant (F138C H221C) was engineered on a background PlyB variant that lacks the wildtype cysteine (C487A) in order to avoid incorrect disulphide bond formation. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : pleurotolysin prepore on liposomes (TMH1 lock) trapped with engin...
Entire | Name: pleurotolysin prepore on liposomes (TMH1 lock) trapped with engineered disulphide |
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Components |
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-Supramolecule #1000: pleurotolysin prepore on liposomes (TMH1 lock) trapped with engin...
Supramolecule | Name: pleurotolysin prepore on liposomes (TMH1 lock) trapped with engineered disulphide type: sample / ID: 1000 Details: 13 fold symmetrical ring oligomers of 26 PlyA and 13 PlyB (C487A, F138C, H221C variant) on liposomes Number unique components: 2 |
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-Macromolecule #1: pleurotolysin A
Macromolecule | Name: pleurotolysin A / type: protein_or_peptide / ID: 1 / Name.synonym: PlyA / Number of copies: 26 / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 17 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | UniProtKB: Pleurotolysin A / GO: hemolysis by symbiont of host erythrocytes / InterPro: Hemolysin, aegerolysin type |
-Macromolecule #2: pleurotolysin B
Macromolecule | Name: pleurotolysin B / type: protein_or_peptide / ID: 2 / Name.synonym: PlyB / Details: C487A, F138C, H221C variant / Number of copies: 13 / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 52 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | UniProtKB: Pleurotolysin B / GO: hemolysis by symbiont of host erythrocytes InterPro: Membrane attack complex component/perforin (MACPF) domain |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.02 mg/mL |
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Buffer | pH: 7.4 / Details: 50 mM NaCl, 20 mM Hepes |
Grid | Details: 300 mesh lacey copper grids |
Vitrification | Cryogen 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
Microscope | FEI POLARA 300 |
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Temperature | Min: 90 K / Max: 102 K / Average: 94 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 115,000 times magnification |
Date | Jul 1, 2011 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 274 / Average electron dose: 25 e/Å2 / Bits/pixel: 16 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Calibrated magnification: 76148 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.3 mm / Nominal defocus max: 3.6 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 59000 |
Sample stage | Specimen holder: liquid nitrogen cooled / Specimen holder model: GATAN HELIUM |
Experimental equipment | ![]() Model: Tecnai Polara / Image courtesy: FEI Company |
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Image processing
Details | 2,400 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. |
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CTF correction | Details: Estimated with CTFFIND3, then phases flipped for each particle |
Final reconstruction | Applied symmetry - Point group: C13 (13 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: OTHER / Software - Name: Imagic, Spider / Details: SPIDER operation BP RP was used for reconstruction / Number images used: 1150 |
-Atomic model buiding 1
Initial model | PDB ID: |
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Software | Name: ![]() |
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation coefficient |
Output model | ![]() PDB-4v3a: |