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- PDB-3nsj: The X-ray crystal structure of lymphocyte perforin -

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Basic information

Entry
Database: PDB / ID: 3nsj
TitleThe X-ray crystal structure of lymphocyte perforin
ComponentsPerforin-1
KeywordsIMMUNE SYSTEM / Pore forming protein
Function / homology
Function and homology information


immune response to tumor cell / granzyme-mediated programmed cell death signaling pathway / cytolytic granule / pore-forming activity / protein transmembrane transport / immunological synapse formation / wide pore channel activity / protein import / positive regulation of killing of cells of another organism / defense response to tumor cell ...immune response to tumor cell / granzyme-mediated programmed cell death signaling pathway / cytolytic granule / pore-forming activity / protein transmembrane transport / immunological synapse formation / wide pore channel activity / protein import / positive regulation of killing of cells of another organism / 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 / membrane / identical protein binding / 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 / Membrane attack complex/perforin (MACPF) domain profile. / MAC/Perforin domain / Membrane attack complex component/perforin (MACPF) domain / C2 domain / C2 domain / Protein kinase C conserved region 2 (CalB) ...Perforin-1, C2 domain / Membrane attack complex component/perforin domain, conserved site / Membrane attack complex/perforin (MACPF) domain signature. / membrane-attack complex / perforin / Membrane attack complex/perforin (MACPF) domain profile. / MAC/Perforin domain / Membrane attack complex component/perforin (MACPF) domain / C2 domain / C2 domain / Protein kinase C conserved region 2 (CalB) / C2 domain / C2 domain profile. / C2 domain superfamily / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
IODIDE ION / Perforin-1
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MIRAS / Resolution: 2.75 Å
AuthorsLaw, R.H. / Whisstock, J.C. / Caradoc-Davies, T.T.
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 1, 2010Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 3, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Jul 29, 2020Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / struct_site / struct_site_gen
Item: _chem_comp.name / _chem_comp.type ..._chem_comp.name / _chem_comp.type / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_ref_seq_dif.details
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 1.3Dec 27, 2023Group: Data collection / Database references / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Perforin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)61,95513
Polymers60,7991
Non-polymers1,15612
Water5,026279
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)78.640, 109.800, 141.030
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

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Protein / Sugars , 2 types, 2 molecules A

#1: Protein Perforin-1 / / P1 / Lymphocyte pore-forming protein / Cytolysin


Mass: 60798.973 Da / Num. of mol.: 1 / Mutation: R213E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Prf1, Pfp / Plasmid: pBacPAK9 / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): Sf21 / References: UniProt: P10820
#6: 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 / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
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

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Non-polymers , 5 types, 290 molecules

#2: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


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


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#4: Chemical
ChemComp-IOD / IODIDE ION / Iodide


Mass: 126.904 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: I
#5: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 279 / Source method: isolated from a natural source / Formula: H2O

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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: 5.01 Å3/Da / Density % sol: 75.44 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 0.5 M sodium acetate, 0.1 M imidazole, pH 6.5, VAPOR DIFFUSION, HANGING DROP, temperature 295K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.953689 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Feb 25, 2010 / Details: Silicon mirrors (adaptive and U-bent)
RadiationMonochromator: Double crystal monochromator (Si111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.953689 Å / Relative weight: 1
ReflectionRedundancy: 12.7 % / Av σ(I) over netI: 3.9 / Number: 205135 / Rsym value: 0.186 / D res high: 3.501 Å / D res low: 87.549 Å / Num. obs: 16169 / % possible obs: 99.5
Diffraction reflection shell
Highest resolution (Å)Lowest resolution (Å)% possible obs (%)IDRmerge(I) obsRsym valueRedundancy
11.0719.8183.610.0650.06511.5
7.8311.0710010.0770.07712.3
6.397.8310010.1150.11512.6
5.536.3910010.1560.15612.5
4.955.5310010.1510.15112.4
4.524.9510010.1610.16112.6
4.184.5210010.1990.19912.8
3.914.1810010.2840.28412.9
3.693.9110010.3970.39712.9
3.53.6910010.5430.54313
ReflectionResolution: 2.75→86.636 Å / Num. all: 32550 / Num. obs: 32550 / % possible obs: 100 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 16.5 % / Biso Wilson estimate: 85.48 Å2 / Rmerge(I) obs: 0.11 / Rsym value: 0.11 / Net I/σ(I): 18.8
Reflection shellResolution: 2.75→2.9 Å / Redundancy: 16.9 % / Rmerge(I) obs: 0.1093 / Mean I/σ(I) obs: 3.1 / Rsym value: 0.1093 / % possible all: 100

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Phasing

PhasingMethod: MIRAS

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Processing

Software
NameVersionClassificationNB
SCALA3.3.9data processing
SHARPphasing
BUSTER-TNTrefinement
PDB_EXTRACT3.1data extraction
ADSCQuantumdata collection
XDSdata reduction
SCALAdata scaling
BUSTER2.8.0refinement
RefinementMethod to determine structure: MIRAS / Resolution: 2.75→47.36 Å / Cor.coef. Fo:Fc: 0.9289 / Cor.coef. Fo:Fc free: 0.9277 / Occupancy max: 1 / Occupancy min: 0.4 / Isotropic thermal model: anisotropic / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.2146 1683 5.2 %RANDOM
Rwork0.1962 ---
obs0.1971 32390 100 %-
all-32550 --
Displacement parametersBiso mean: 80.51 Å2
Baniso -1Baniso -2Baniso -3
1-13.2715 Å20 Å20 Å2
2---12.4774 Å20 Å2
3----0.7942 Å2
Refine analyzeLuzzati coordinate error obs: 0.386 Å
Refinement stepCycle: LAST / Resolution: 2.75→47.36 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4036 0 40 279 4355
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0074198HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.985716HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d1383SINUSOIDAL2
X-RAY DIFFRACTIONt_incorr_chiral_ct
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_trig_c_planes89HARMONIC2
X-RAY DIFFRACTIONt_gen_planes638HARMONIC5
X-RAY DIFFRACTIONt_it4198HARMONIC20
X-RAY DIFFRACTIONt_nbd2SEMIHARMONIC5
X-RAY DIFFRACTIONt_omega_torsion2.28
X-RAY DIFFRACTIONt_other_torsion17.36
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_chiral_improper_torsion
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact
LS refinement shellResolution: 2.75→2.84 Å / Total num. of bins used: 16
RfactorNum. reflection% reflection
Rfree0.2758 162 5.65 %
Rwork0.2307 2707 -
all0.2333 2869 -
Refinement TLS params.Method: refined / Origin x: 10.7897 Å / Origin y: 16.6897 Å / Origin z: 92.7071 Å
111213212223313233
T-0.0798 Å20.0459 Å2-0.0011 Å2-0.0328 Å20.0267 Å2---0.0264 Å2
L0.4139 °2-0.5791 °20.7771 °2-0.7434 °2-1.0808 °2--1.2877 °2
S-0.0139 Å °-0.1102 Å °0.0365 Å °0.0306 Å °0.0694 Å °0.0351 Å °-0.0121 Å °0.0039 Å °-0.0555 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ A|21 - A|135 A|137 - A|551 A|652 - A|654 A|701 - A|702 A|710 - A|713 A|720 - A|721 A|901 - A|901 }A21 - 135
2X-RAY DIFFRACTION1{ A|21 - A|135 A|137 - A|551 A|652 - A|654 A|701 - A|702 A|710 - A|713 A|720 - A|721 A|901 - A|901 }A137 - 551
3X-RAY DIFFRACTION1{ A|21 - A|135 A|137 - A|551 A|652 - A|654 A|701 - A|702 A|710 - A|713 A|720 - A|721 A|901 - A|901 }A652 - 654
4X-RAY DIFFRACTION1{ A|21 - A|135 A|137 - A|551 A|652 - A|654 A|701 - A|702 A|710 - A|713 A|720 - A|721 A|901 - A|901 }A701 - 702
5X-RAY DIFFRACTION1{ A|21 - A|135 A|137 - A|551 A|652 - A|654 A|701 - A|702 A|710 - A|713 A|720 - A|721 A|901 - A|901 }A710 - 713
6X-RAY DIFFRACTION1{ A|21 - A|135 A|137 - A|551 A|652 - A|654 A|701 - A|702 A|710 - A|713 A|720 - A|721 A|901 - A|901 }A720 - 721
7X-RAY DIFFRACTION1{ A|21 - A|135 A|137 - A|551 A|652 - A|654 A|701 - A|702 A|710 - A|713 A|720 - A|721 A|901 - A|901 }A901

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