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- EMDB-1769: Perforin Pore -

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Basic information

Entry
Database: EMDB / ID: EMD-1769
TitlePerforin Pore
Map datacryo EM reconstruction of perforin pore with 20 fold symmetry (Related to EM entries 1772 and 1773)
Sample
  • Sample: Perforin pores on liposomes
  • Protein or peptide: PerforinPerforin-1
KeywordsMACPF-CDC superfamily / pore-forming proteins
Function / homologyMembrane attack complex component/perforin (MACPF) domain
Function and homology information
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 28.5 Å
AuthorsLukoyanova 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
CitationJournal: 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
DepositionJul 27, 2010-
Header (metadata) releaseSep 24, 2010-
Map releaseNov 3, 2010-
UpdateNov 16, 2010-
Current statusNov 16, 2010Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.007
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.007
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1769image.png

Downloads & links

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Map

FileDownload / File: emd_1769.map.gz / Format: CCP4 / Size: 12.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcryo EM reconstruction of perforin pore with 20 fold symmetry (Related to EM entries 1772 and 1773)
Voxel sizeX=Y=Z: 2.2 Å
Density
Contour LevelBy AUTHOR: 0.007 / Movie #1: 0.007
Minimum - Maximum-0.0255626 - 0.0517069
Average (Standard dev.)0.00199938 (±0.00765263)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 330 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.22.22.2
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z330.000330.000330.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS150150150
D min/max/mean-0.0260.0520.002

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Supplemental data

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Sample components

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Entire : Perforin pores on liposomes

EntireName: Perforin pores on liposomes
Components
  • Sample: Perforin pores on liposomes
  • Protein or peptide: PerforinPerforin-1

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Supramolecule #1000: Perforin pores on liposomes

SupramoleculeName: 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

MacromoleculeName: Perforin / type: protein_or_peptide / ID: 1 / Name.synonym: Perforin / Number of copies: 20 / Oligomeric state: 20-mer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceInterPro: Membrane attack complex component/perforin (MACPF) domain

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.005 mg/mL
BufferpH: 8 / Details: 0.15M NaCl, 1mM CaCl2, 20mM Hepes
GridDetails: 300, 400 lacey copper grids
VitrificationCryogen 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

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated 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 stageSpecimen holder: Side entry LN2 cooled, single tilt / Specimen holder model: GATAN LIQUID NITROGEN
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000x magnification
DateApr 23, 2007
Image recordingCategory: 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

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Image processing

CTF correctionDetails: Estimated with CTFFIND3, then phases flipped for each particle
Final two d classificationNumber classes: 10
Final reconstructionApplied 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
DetailsImage regions containing the pores with small surrounding areas of membrane were selected manually using EMAN-Boxer software

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Atomic model buiding 1

Initial modelPDB ID:

3nsj

SoftwareName: UCSF Chimera
Detailsfitting was done manually
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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