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- PDB-6sb1: Crystal structure of murine perforin-2 P2 domain crystal form 1 -

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Basic information

Entry
Database: PDB / ID: 6sb1
TitleCrystal structure of murine perforin-2 P2 domain crystal form 1
ComponentsMacrophage-expressed gene 1 protein
KeywordsTOXIN / Pore-forming protein / perforin / 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 / 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)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SIRAS / Resolution: 2.05 Å
AuthorsNi, T. / Ginger, L. / 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
Revision 1.1Feb 26, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Macrophage-expressed gene 1 protein
B: Macrophage-expressed gene 1 protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)64,9036
Polymers64,5912
Non-polymers3124
Water1,63991
1
A: Macrophage-expressed gene 1 protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)32,5154
Polymers32,2961
Non-polymers2203
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Macrophage-expressed gene 1 protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)32,3882
Polymers32,2961
Non-polymers921
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)70.530, 31.270, 103.650
Angle α, β, γ (deg.)90.000, 91.420, 90.000
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and (resid 376 through 538 or resid 549 through 579))
21(chain B and resid 376 through 579)

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111(chain A and (resid 376 through 538 or resid 549 through 579))A376 - 538
121(chain A and (resid 376 through 538 or resid 549 through 579))A549 - 579
211(chain B and resid 376 through 579)B376 - 579

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Components

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


Mass: 32295.625 Da / Num. of mol.: 2
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: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 91 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.67 Å3/Da / Density % sol: 53.99 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / Details: 0.1M MES pH6.0, 10% PEG6000, 4mM L-cysteine

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I24 / Wavelength: 0.96861 Å
DetectorType: DECTRIS PILATUS3 X 6M / Detector: PIXEL / Date: Jan 23, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.96861 Å / Relative weight: 1
ReflectionResolution: 2.05→58.98 Å / Num. obs: 28963 / % possible obs: 99.4 % / Redundancy: 6.5 % / Net I/σ(I): 12.7
Reflection shellResolution: 2.05→2.09 Å / Num. unique obs: 7980

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Processing

Software
NameVersionClassification
PHENIXdev_2645refinement
PDB_EXTRACT3.25data extraction
xia2data reduction
xia2data scaling
PHENIXphasing
RefinementMethod to determine structure: SIRAS / Resolution: 2.05→57.632 Å / SU ML: 0.26 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 29.3
RfactorNum. reflection% reflection
Rfree0.2428 1432 4.95 %
Rwork0.212 --
obs0.2135 28948 99.31 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 205.43 Å2 / Biso mean: 77.006 Å2 / Biso min: 31.09 Å2
Refinement stepCycle: final / Resolution: 2.05→57.632 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2988 0 19 91 3098
Biso mean--79.31 68.9 -
Num. residues----392
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0093089
X-RAY DIFFRACTIONf_angle_d1.0934181
X-RAY DIFFRACTIONf_chiral_restr0.061455
X-RAY DIFFRACTIONf_plane_restr0.007533
X-RAY DIFFRACTIONf_dihedral_angle_d15.0331851
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A1815X-RAY DIFFRACTION9.162TORSIONAL
12B1815X-RAY DIFFRACTION9.162TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.05-2.12330.37411420.328270899
2.1233-2.20830.30611300.2916275299
2.2083-2.30880.29141380.276268699
2.3088-2.43050.27841600.2426272099
2.4305-2.58280.29071410.2547273499
2.5828-2.78220.31231650.2574269799
2.7822-3.06220.27181200.23362777100
3.0622-3.50520.23931460.2052784100
3.5052-4.4160.23931460.1865277599
4.416-51.810.19211440.1843288399
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
18.93515.7192-2.66887.7789-2.41254.74130.5436-0.5691.06420.4819-0.21090.6261-0.44350.2603-0.26450.53990.02240.09710.2396-0.05790.3989-0.103728.573360.446
27.1785-1.3464-4.8391.64041.03715.0903-0.2281-0.2395-0.21340.4018-0.06990.67290.2301-0.33390.21550.6934-0.01640.2290.3983-0.07030.6817-15.970121.787569.6138
38.56921.98550.10698.73931.65815.6067-0.3202-1.0113-0.5060.58470.2395-1.44240.00520.88360.0660.50640.0905-0.16210.49330.04490.548814.142722.277463.308
46.2694-2.67170.83668.99241.62942.75560.49140.10730.6914-0.2629-0.27460.1075-0.4991-0.0865-0.18450.518-0.01350.06010.23620.03940.352629.287213.005695.6421
57.6177-0.1322-5.3492.1178-0.14725.2744-0.42130.0526-0.4311-0.3465-0.1028-0.44560.25230.3780.42120.5817-0.01870.14250.36230.06940.549445.75476.158785.4148
62.2406-2.9493.17336.743-6.57286.49980.1098-1.70841.02523.89941.59423.9122-3.6943-3.7614-1.35971.68840.50360.46031.2469-0.03521.61813.747317.8783106.2795
77.0384-0.4231-0.61857.3290.38636.1917-0.54871.2059-0.6382-0.39120.08741.5920.2985-1.00850.37480.4858-0.1419-0.08690.6311-0.13870.788916.16146.150692.3161
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 376 through 412 )A376 - 412
2X-RAY DIFFRACTION2chain 'A' and (resid 413 through 464 )A413 - 464
3X-RAY DIFFRACTION3chain 'A' and (resid 465 through 579 )A465 - 579
4X-RAY DIFFRACTION4chain 'B' and (resid 374 through 412 )B374 - 412
5X-RAY DIFFRACTION5chain 'B' and (resid 413 through 464 )B413 - 464
6X-RAY DIFFRACTION6chain 'B' and (resid 465 through 474 )B465 - 474
7X-RAY DIFFRACTION7chain 'B' and (resid 475 through 579 )B475 - 579

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