+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-1769 | |||||||||
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Title | Perforin Pore | |||||||||
Map data | cryo EM reconstruction of perforin pore with 20 fold symmetry (Related to EM entries 1772 and 1773) | |||||||||
Sample |
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Keywords | MACPF-CDC superfamily / pore-forming proteins | |||||||||
Function / homology | Membrane attack complex component/perforin (MACPF) domain Function and homology information | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 28.5 Å | |||||||||
Authors | Lukoyanova N / Law RHP / Voskoboinik I / Caradoc-Davies TT / Baran K / Dunstone MA / D'Angelo ME / Orlova EV / Coulibaly F / Verschoor S ...Lukoyanova N / Law RHP / Voskoboinik I / Caradoc-Davies TT / Baran K / Dunstone MA / D'Angelo ME / Orlova EV / Coulibaly F / Verschoor S / Browne KA / Ciccone A / Kuiper MJ / Bird PI / Trapani JA / Whisstock JC / Saibil HR | |||||||||
Citation | Journal: Nature / Year: 2010 Title: The structural basis for membrane binding and pore formation by lymphocyte perforin. Authors: Ruby H P Law / Natalya Lukoyanova / Ilia Voskoboinik / Tom T Caradoc-Davies / Katherine Baran / Michelle A Dunstone / Michael E D'Angelo / Elena V Orlova / Fasséli Coulibaly / Sandra ...Authors: Ruby H P Law / Natalya Lukoyanova / Ilia Voskoboinik / Tom T Caradoc-Davies / Katherine Baran / Michelle A Dunstone / Michael E D'Angelo / Elena V Orlova / Fasséli Coulibaly / Sandra Verschoor / Kylie A Browne / Annette Ciccone / Michael J Kuiper / Phillip I Bird / Joseph A Trapani / Helen R Saibil / James C Whisstock / Abstract: Natural killer cells and cytotoxic T lymphocytes accomplish the critically important function of killing virus-infected and neoplastic cells. They do this by releasing the pore-forming protein ...Natural killer cells and cytotoxic T lymphocytes accomplish the critically important function of killing virus-infected and neoplastic cells. They do this by releasing the pore-forming protein perforin and granzyme proteases from cytoplasmic granules into the cleft formed between the abutting killer and target cell membranes. Perforin, a 67-kilodalton multidomain protein, oligomerizes to form pores that deliver the pro-apoptopic granzymes into the cytosol of the target cell. The importance of perforin is highlighted by the fatal consequences of congenital perforin deficiency, with more than 50 different perforin mutations linked to familial haemophagocytic lymphohistiocytosis (type 2 FHL). Here we elucidate the mechanism of perforin pore formation by determining the X-ray crystal structure of monomeric murine perforin, together with a cryo-electron microscopy reconstruction of the entire perforin pore. Perforin is a thin 'key-shaped' molecule, comprising an amino-terminal membrane attack complex perforin-like (MACPF)/cholesterol dependent cytolysin (CDC) domain followed by an epidermal growth factor (EGF) domain that, together with the extreme carboxy-terminal sequence, forms a central shelf-like structure. A C-terminal C2 domain mediates initial, Ca(2+)-dependent membrane binding. Most unexpectedly, however, electron microscopy reveals that the orientation of the perforin MACPF domain in the pore is inside-out relative to the subunit arrangement in CDCs. These data reveal remarkable flexibility in the mechanism of action of the conserved MACPF/CDC fold and provide new insights into how related immune defence molecules such as complement proteins assemble into pores. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_1769.map.gz | 12.3 MB | EMDB map data format | |
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Header (meta data) | emd-1769-v30.xml emd-1769.xml | 10.9 KB 10.9 KB | Display Display | EMDB header |
Images | 1769image.png | 113.8 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-1769 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-1769 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_1769.map.gz / Format: CCP4 / Size: 12.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | cryo EM reconstruction of perforin pore with 20 fold symmetry (Related to EM entries 1772 and 1773) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.2 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Perforin pores on liposomes
Entire | Name: Perforin pores on liposomes |
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Components |
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-Supramolecule #1000: Perforin pores on liposomes
Supramolecule | Name: Perforin pores on liposomes / type: sample / ID: 1000 Details: Pores heterogeneous in size (15-25 nm) and symmetry (18-28 subunits) Oligomeric state: 20-mer / Number unique components: 1 |
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-Macromolecule #1: Perforin
Macromolecule | Name: Perforin / type: protein_or_peptide / ID: 1 / Name.synonym: Perforin / Number of copies: 20 / Oligomeric state: 20-mer / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human |
Recombinant expression | Organism: Spodoptera frugiperda (fall armyworm) |
Sequence | InterPro: Membrane attack complex component/perforin (MACPF) domain |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.005 mg/mL |
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Buffer | pH: 8 / Details: 0.15M NaCl, 1mM CaCl2, 20mM Hepes |
Grid | Details: 300, 400 lacey copper grids |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: OTHER / Details: Vitrification instrument: Vitrobot Method: liposomes were applied to neg. glow discharged grids first, then protein solution was added. Grids were left inside Vitrobot equilibrated at 37C for 1-2 min for pore formaition. Blot for 2 seconds before plunging |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 2.424 µm / Nominal defocus min: 0.807 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder: Side entry LN2 cooled, single tilt / Specimen holder model: GATAN LIQUID NITROGEN |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 150,000x magnification |
Date | Apr 23, 2007 |
Image recording | Category: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 260 / Average electron dose: 20 e/Å2 |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: Estimated with CTFFIND3, then phases flipped for each particle |
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Final two d classification | Number classes: 10 |
Final reconstruction | Applied symmetry - Point group: C20 (20 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 28.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Imagic, Spider Details: 20-fold symmetrised reconstruction. Hand of the map has not been determined Number images used: 59 |
Details | Image regions containing the pores with small surrounding areas of membrane were selected manually using EMAN-Boxer software |
-Atomic model buiding 1
Initial model | PDB ID: |
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Software | Name: UCSF Chimera |
Details | fitting was done manually |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |