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- PDB-6sb3: CryoEM structure of murine perforin-2 ectodomain in a pre-pore form -

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Basic information

Entry
Database: PDB / ID: 6sb3
TitleCryoEM structure of murine perforin-2 ectodomain in a pre-pore form
ComponentsMacrophage-expressed gene 1 protein
KeywordsTOXIN / pore-forming protein / pre-pore / MACPF
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 / MAC/Perforin domain / Membrane attack complex/perforin (MACPF) domain profile. / Membrane attack complex component/perforin (MACPF) domain
Similarity search - Domain/homology
Macrophage-expressed gene 1 protein
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsNi, T. / Yu, X. / Gilbert, R.J.C.
Funding support United Kingdom, 1items
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, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 5, 2020Provider: repository / Type: Initial release
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Revision 1.2Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
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Structure visualization

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Assembly

Deposited unit
A: Macrophage-expressed gene 1 protein
B: Macrophage-expressed gene 1 protein
C: Macrophage-expressed gene 1 protein
D: Macrophage-expressed gene 1 protein
E: Macrophage-expressed gene 1 protein
F: Macrophage-expressed gene 1 protein
G: Macrophage-expressed gene 1 protein
H: Macrophage-expressed gene 1 protein
I: Macrophage-expressed gene 1 protein
J: Macrophage-expressed gene 1 protein
K: Macrophage-expressed gene 1 protein
L: Macrophage-expressed gene 1 protein
M: Macrophage-expressed gene 1 protein
N: Macrophage-expressed gene 1 protein
O: Macrophage-expressed gene 1 protein
P: Macrophage-expressed gene 1 protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,145,15148
Polymers1,138,07216
Non-polymers7,07932
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area141870 Å2
ΔGint-343 kcal/mol
Surface area328870 Å2
MethodPISA

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Components

#1: Protein
Macrophage-expressed gene 1 protein / Mpg-1 / Perforin-2 / P-2 / Protein MPS1


Mass: 71129.531 Da / Num. of mol.: 16
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Mpeg1 / Production host: Homo sapiens (human) / References: UniProt: A1L314
#2: Sugar...
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 32
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Murine perforin-2 ecto domain / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293T / Plasmid: pHLsec-1D4
Buffer solutionpH: 5.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Homemade
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Alignment procedure: COMA FREE
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: dev_3488: / Classification: refinement
EM software
IDNameCategory
1RELIONparticle selection
2SerialEMimage acquisition
4GctfCTF correction
8PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C16 (16 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 41693 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00277344
ELECTRON MICROSCOPYf_angle_d0.552105104
ELECTRON MICROSCOPYf_dihedral_angle_d16.39246704
ELECTRON MICROSCOPYf_chiral_restr0.04112160
ELECTRON MICROSCOPYf_plane_restr0.00413520

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