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- EMDB-13269: The pore conformation of lymphocyte perforin -

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Basic information

Entry
Database: EMDB / ID: EMD-13269
TitleThe pore conformation of lymphocyte perforin
Map data
Sample
  • Complex: Oligomerised mouse Perforin in its inserted state
    • Protein or peptide: Perforin-1
  • Ligand: CALCIUM ION
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


immune response to tumor cell / granzyme-mediated programmed cell death signaling pathway / cytolytic granule / pore-forming activity / immunological synapse formation / protein transmembrane transport / wide pore channel activity / positive regulation of killing of cells of another organism / protein import / defense response to tumor cell ...immune response to tumor cell / granzyme-mediated programmed cell death signaling pathway / cytolytic granule / pore-forming activity / immunological synapse formation / protein transmembrane transport / wide pore channel activity / positive regulation of killing of cells of another organism / protein import / defense response to tumor cell / immunological synapse / protein secretion / endosome lumen / protein homooligomerization / T cell mediated cytotoxicity / circadian rhythm / cytoplasmic vesicle / defense response to virus / killing of cells of another organism / calcium ion binding / extracellular space / identical protein binding / membrane / plasma membrane / cytosol
Similarity search - Function
Perforin-1, C2 domain / : / Membrane attack complex component/perforin domain, conserved site / Membrane attack complex/perforin (MACPF) domain signature. / membrane-attack complex / perforin / MAC/Perforin domain / Membrane attack complex/perforin (MACPF) domain profile. / Membrane attack complex component/perforin (MACPF) domain / Protein kinase C conserved region 2 (CalB) / C2 domain ...Perforin-1, C2 domain / : / Membrane attack complex component/perforin domain, conserved site / Membrane attack complex/perforin (MACPF) domain signature. / membrane-attack complex / perforin / MAC/Perforin domain / Membrane attack complex/perforin (MACPF) domain profile. / Membrane attack complex component/perforin (MACPF) domain / Protein kinase C conserved region 2 (CalB) / C2 domain / C2 domain / C2 domain profile. / C2 domain superfamily
Similarity search - Domain/homology
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsIvanova ME / Lukoyanova N / Malhotra S / Topf M / Trapani JA / Voskoboinik I / Saibil HR
Funding support United Kingdom, 6 items
OrganizationGrant numberCountry
Wellcome Trust106249/Z/14/Z United Kingdom
Wellcome Trust209250/Z/17/Z United Kingdom
Wellcome Trust208398/Z/17/Z United Kingdom
European Research Council (ERC)294408 United Kingdom
Wellcome Trust202679/Z/16/Z United Kingdom
Wellcome Trust206166/Z/17/Z United Kingdom
CitationJournal: Sci Adv / Year: 2022
Title: The pore conformation of lymphocyte perforin.
Authors: Marina E Ivanova / Natalya Lukoyanova / Sony Malhotra / Maya Topf / Joseph A Trapani / Ilia Voskoboinik / Helen R Saibil /
Abstract: Perforin is a pore-forming protein that facilitates rapid killing of pathogen-infected or cancerous cells by the immune system. Perforin is released from cytotoxic lymphocytes, together with ...Perforin is a pore-forming protein that facilitates rapid killing of pathogen-infected or cancerous cells by the immune system. Perforin is released from cytotoxic lymphocytes, together with proapoptotic granzymes, to bind to a target cell membrane where it oligomerizes and forms pores. The pores allow granzyme entry, which rapidly triggers the apoptotic death of the target cell. Here, we present a 4-Å resolution cryo-electron microscopy structure of the perforin pore, revealing previously unidentified inter- and intramolecular interactions stabilizing the assembly. During pore formation, the helix-turn-helix motif moves away from the bend in the central β sheet to form an intermolecular contact. Cryo-electron tomography shows that prepores form on the membrane surface with minimal conformational changes. Our findings suggest the sequence of conformational changes underlying oligomerization and membrane insertion, and explain how several pathogenic mutations affect function.
History
DepositionJul 29, 2021-
Header (metadata) releaseFeb 16, 2022-
Map releaseFeb 16, 2022-
UpdateFeb 23, 2022-
Current statusFeb 23, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.024
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.024
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7pag
  • Surface level: 0.024
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7pag
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13269.png

Downloads & links

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Map

FileDownload / File: emd_13269.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.34 Å/pix.
x 240 pix.
= 321.6 Å		1.34 Å/pix.
x 240 pix.
= 321.6 Å		1.34 Å/pix.
x 240 pix.
= 321.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.34 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.024 / Movie #1: 0.024
Minimum - Maximum-0.079215944 - 0.1557281
Average (Standard dev.)0.00030226613 (±0.0036413132)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 321.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.341.341.34
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z321.600321.600321.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0790.1560.000

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Supplemental data

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Sample components

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Entire : Oligomerised mouse Perforin in its inserted state

EntireName: Oligomerised mouse Perforin in its inserted state
Components
  • Complex: Oligomerised mouse Perforin in its inserted state
    • Protein or peptide: Perforin-1
  • Ligand: CALCIUM ION
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Oligomerised mouse Perforin in its inserted state

SupramoleculeName: Oligomerised mouse Perforin in its inserted state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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Macromolecule #1: Perforin-1

MacromoleculeName: Perforin-1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 60.827051 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: PCYTATRSEC KQKHKFVPGV WMAGEGMDVT TLRRSGSFPV NTQRFLRPDR TCTLCKNSLM RDATQRLPVA ITHWRPHSSH CQRNVAAAK VHSTEGVARE AAANINNDWR VGLDVNPRPE ANMRASVAGS HSKVANFAAE KTYQDQYNFN SDTVECRMYS F RLVQKPPL ...String:
PCYTATRSEC KQKHKFVPGV WMAGEGMDVT TLRRSGSFPV NTQRFLRPDR TCTLCKNSLM RDATQRLPVA ITHWRPHSSH CQRNVAAAK VHSTEGVARE AAANINNDWR VGLDVNPRPE ANMRASVAGS HSKVANFAAE KTYQDQYNFN SDTVECRMYS F RLVQKPPL HLDFKKALRA LPRNFNSSTE HAYHRLISSY GTHFITAVDL GGRISVLTAL RTCQLTLNGL TADEVGDCLN VE AQVSIGA QASVSSEYKA CEEKKKQHKM ATSFHQTYRE RHVEVLGGPL DSTHDLLFGN QATPEQFSTW TASLPSNPGL VDY SLEPLH TLLEEQNPKR EALRQAISHY IMSRARWQNC SRPCRSGQHK SSHDSCQCEC QDSKVTNQDC CPRQRGLAHL VVSN FRAEH LWGDYTTATD AYLKVFFGGQ EFRTGVVWNN NNPRWTDKMD FENVLLSTGG PLRVQVWDAD YGWDDDLLGS CDRSP HSGF HEVTCELNHG RVKFSYHAKC LPHLTGGTCL EYAPQGLLGD PPGNRSGAVW HHHHHH

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 3 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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

BufferpH: 7.5
GridModel: UltrAuFoil R2/2 / Material: GOLD / Support film - Material: GRAPHENE OXIDE / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 49.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF
Details: 229,789 particles were symmetry expanded with C22 symmetry giving the final number of particles 5,055,358
Number images used: 229789
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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