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- EMDB-10134: CryoEM structure of murine perforin-2 ectodomain in a pre-pore form -

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Basic information

Entry
Database: EMDB / ID: EMD-10134
TitleCryoEM structure of murine perforin-2 ectodomain in a pre-pore form
Map data
Sample
  • Complex: Murine perforin-2 ecto domain
    • Protein or peptide: Macrophage-expressed gene 1 protein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


dendritic cell antigen processing and presentation / antigen processing and presentation of exogenous peptide antigen / phagolysosome membrane / endolysosome / pore-forming activity / antibacterial innate immune response / wide pore channel activity / antigen processing and presentation of exogenous peptide antigen via MHC class I / phagocytic vesicle / phagocytic vesicle membrane ...dendritic cell antigen processing and presentation / antigen processing and presentation of exogenous peptide antigen / phagolysosome membrane / endolysosome / pore-forming activity / antibacterial innate immune response / wide pore channel activity / antigen processing and presentation of exogenous peptide antigen via MHC class I / phagocytic vesicle / phagocytic vesicle membrane / cytoplasmic vesicle / defense response to Gram-negative bacterium / adaptive immune response / defense response to Gram-positive bacterium / defense response to bacterium
Similarity search - Function
Macrophage-expressed gene 1 protein / membrane-attack complex / perforin / Membrane attack complex/perforin (MACPF) domain profile. / MAC/Perforin domain / Membrane attack complex component/perforin (MACPF) domain
Similarity search - Domain/homology
Macrophage-expressed gene 1 protein
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsNi T / Yu X / Gilbert RJC
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MR/N000331/1 United Kingdom
CitationJournal: Sci Adv / Year: 2020
Title: Structure and mechanism of bactericidal mammalian perforin-2, an ancient agent of innate immunity.
Authors: Tao Ni / Fang Jiao / Xiulian Yu / Saša Aden / Lucy Ginger / Sophie I Williams / Fangfang Bai / Vojtěch Pražák / Dimple Karia / Phillip Stansfeld / Peijun Zhang / George Munson / Gregor ...Authors: Tao Ni / Fang Jiao / Xiulian Yu / Saša Aden / Lucy Ginger / Sophie I Williams / Fangfang Bai / Vojtěch Pražák / Dimple Karia / Phillip Stansfeld / Peijun Zhang / George Munson / Gregor Anderluh / Simon Scheuring / Robert J C Gilbert /
Abstract: Perforin-2 (MPEG1) is thought to enable the killing of invading microbes engulfed by macrophages and other phagocytes, forming pores in their membranes. Loss of perforin-2 renders individual ...Perforin-2 (MPEG1) is thought to enable the killing of invading microbes engulfed by macrophages and other phagocytes, forming pores in their membranes. Loss of perforin-2 renders individual phagocytes and whole organisms significantly more susceptible to bacterial pathogens. Here, we reveal the mechanism of perforin-2 activation and activity using atomic structures of pre-pore and pore assemblies, high-speed atomic force microscopy, and functional assays. Perforin-2 forms a pre-pore assembly in which its pore-forming domain points in the opposite direction to its membrane-targeting domain. Acidification then triggers pore formation, via a 180° conformational change. This novel and unexpected mechanism prevents premature bactericidal attack and may have played a key role in the evolution of all perforin family proteins.
History
DepositionJul 18, 2019-
Header (metadata) releaseFeb 5, 2020-
Map releaseFeb 5, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface level: 0.02
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  • Surface view with fitted model
  • Atomic models: PDB-6sb3
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10134.png

Downloads & links

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Map

FileDownload / File: emd_10134.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.04 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.116871566 - 0.14980699
Average (Standard dev.)0.0002616061 (±0.0042559784)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 374.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.041.041.04
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z374.400374.400374.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.1170.1500.000

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Supplemental data

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Mask #1

Fileemd_10134_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_10134_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_10134_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Murine perforin-2 ecto domain

EntireName: Murine perforin-2 ecto domain
Components
  • Complex: Murine perforin-2 ecto domain
    • Protein or peptide: Macrophage-expressed gene 1 protein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Murine perforin-2 ecto domain

SupramoleculeName: Murine perforin-2 ecto domain / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293T / Recombinant plasmid: pHLsec-1D4

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Macromolecule #1: Macrophage-expressed gene 1 protein

MacromoleculeName: Macrophage-expressed gene 1 protein / type: protein_or_peptide / ID: 1 / Number of copies: 16 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 71.129531 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: ETGDKPLGET GTTGFQICKN ALKLPVLEVL PGGGWDNLRN VDMGRVMDLT YTNCKTTEDG QYIIPDEVYT IPQKESNLEM NSEVLESWM NYQSTTSLSI NTELALFSRV NGKFSTEFQR MKTLQVKDQA VTTRVQVRNR IYTVKTTPTS ELSLGFTKAL M DICDQLEK ...String:
ETGDKPLGET GTTGFQICKN ALKLPVLEVL PGGGWDNLRN VDMGRVMDLT YTNCKTTEDG QYIIPDEVYT IPQKESNLEM NSEVLESWM NYQSTTSLSI NTELALFSRV NGKFSTEFQR MKTLQVKDQA VTTRVQVRNR IYTVKTTPTS ELSLGFTKAL M DICDQLEK NQTKMATYLA ELLILNYGTH VITSVDAGAA LVQEDHVRSS FLLDNQNSQN TVTASAGIAF LNIVNFKVET DY ISQTSLT KDYLSNRTNS RVQSFGGVPF YPGITLETWQ KGITNHLVAI DRAGLPLHFF IKPDKLPGLP GPLVKKLSKT VET AVRHYY TFNTHPGCTN VDSPNFNFQA NMDDDSCDAK VTNFTFGGVY QECTELSGDV LCQNLEQKNL LTGDFSCPPG YSPV HLLSQ THEEGYSRLE CKKKCTLKIF CKTVCEDVFR VAKAEFRAYW CVAAGQVPDN SGLLFGGVFT DKTINPMTNA QSCPA GYIP LNLFESLKVC VSLDYELGFK FSVPFGGFFS CIMGNPLVNS DTAKDVRAPS LKKCPGGFSQ HLAVISDGCQ VSYCVK AGI FTGGSLLPVR LPPYTKPPLM SQVATNTVIV TNSETARSWI KDPQTNQWKL GEPLELRRAM TVIHGDSNGM SGGGTET SQ VAPA

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

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 2 / Number of copies: 32 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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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: 5.5
GridModel: Homemade / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C16 (16 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 41693
FSC plot (resolution estimation)

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