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- EMDB-1772: Perforin monomer, conformation 1 -

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Basic information

Entry
Database: EMDB / ID: EMD-1772
TitlePerforin monomer, conformation 1
Map datanegative stain reconstruction of perforin monomer. (Related to EMDB entries 1773 and 1769)
Sample
  • Sample: PerforinPerforin-1
  • Protein or peptide: PerforinPerforin-1
KeywordsMACPF-CDC superfamily / pore-forming proteins
Function / homologyMembrane attack complex component/perforin (MACPF) domain
Function and homology information
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 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
DepositionAug 3, 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: 0.008
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.008
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1772image.png

Downloads & links

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Map

FileDownload / File: emd_1772.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationnegative stain reconstruction of perforin monomer. (Related to EMDB entries 1773 and 1769)
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.008 / Movie #1: 0.008
Minimum - Maximum-0.0202175 - 0.144454
Average (Standard dev.)0.000243602 (±0.00399893)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 270 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z270.000270.000270.000
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/NS200200200
D min/max/mean-0.0200.1440.000

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Supplemental data

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Sample components

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Entire : Perforin

EntireName: PerforinPerforin-1
Components
  • Sample: PerforinPerforin-1
  • Protein or peptide: PerforinPerforin-1

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Supramolecule #1000: Perforin

SupramoleculeName: Perforin / type: sample / ID: 1000 / Details: Selected one of the multiple conformations / Oligomeric state: Monomer / Number unique components: 1
Molecular weightExperimental: 67.2 KDa / Theoretical: 67.2 KDa

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Macromolecule #1: Perforin

MacromoleculeName: Perforin / type: protein_or_peptide / ID: 1 / Name.synonym: Perforin / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: Mouse
Molecular weightExperimental: 67.2 KDa / Theoretical: 67.2 KDa
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

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 8 / Details: 0.15M NaCl, 1mM CaCl2, 20mM Hepes
StainingType: NEGATIVE
Details: 1% w/v uranyl acetate for 10 sec at room temperature
GridDetails: Home made continuous carbon on 400 mesh copper grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 0.954 µm / Nominal defocus min: 0.506 µm / Nominal magnification: 52000
Sample stageSpecimen holder: Side entry single tilt / Specimen holder model: SIDE ENTRY, EUCENTRIC
Alignment procedureLegacy - Astigmatism: At the specimen level
DateMay 18, 2007
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 32 / Average electron dose: 20 e/Å2 / Bits/pixel: 8

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Image processing

CTF correctionDetails: Estimated with CTFFIND3, then phases flipped for each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Imagic, Spider / Details: The hand of the map has not been determined / Number images used: 3400
DetailsParticles 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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