[English] 日本語
Yorodumi
- EMDB-1769: Perforin Pore -

+
Open data


ID or keywords:

Loading...

-
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: Perforin
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

-
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

-
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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.2 Å/pix.
x 150 pix.
= 330. Å		2.2 Å/pix.
x 150 pix.
= 330. Å		2.2 Å/pix.
x 150 pix.
= 330. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.2 Å
Density

Histogram

Histogram (log scale)
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

-
Supplemental data

-
Sample components

-
Entire : Perforin pores on liposomes

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

-
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

-
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

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
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

-
Electron microscopy

MicroscopeFEI TECNAI F20
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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

-
Image processing

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

-
Atomic model buiding 1

Initial modelPDB ID:

3nsj

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

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more