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- PDB-5nj6: Crystal structure of a thermostabilised human protease-activated ... -

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Basic information

Entry
Database: PDB / ID: 5nj6
TitleCrystal structure of a thermostabilised human protease-activated receptor-2 (PAR2) in ternary complex with Fab3949 and AZ7188 at 4.0 angstrom resolution
Components
  • Fab3949 H
  • Fab3949 L
  • Proteinase-activated receptor 2,Soluble cytochrome b562,Proteinase-activated receptor 2
KeywordsMEMBRANE PROTEIN / GPCR / 7TM
Function / homology
Function and homology information


positive regulation of neutrophil mediated killing of gram-negative bacterium / potassium channel activating, G protein-coupled receptor signaling pathway / positive regulation of renin secretion into blood stream / positive regulation of eosinophil degranulation / leukocyte proliferation / mature conventional dendritic cell differentiation / regulation of chemokine (C-X-C motif) ligand 2 production / positive regulation of glomerular filtration / negative regulation of toll-like receptor 3 signaling pathway / positive regulation of toll-like receptor 3 signaling pathway ...positive regulation of neutrophil mediated killing of gram-negative bacterium / potassium channel activating, G protein-coupled receptor signaling pathway / positive regulation of renin secretion into blood stream / positive regulation of eosinophil degranulation / leukocyte proliferation / mature conventional dendritic cell differentiation / regulation of chemokine (C-X-C motif) ligand 2 production / positive regulation of glomerular filtration / negative regulation of toll-like receptor 3 signaling pathway / positive regulation of toll-like receptor 3 signaling pathway / thrombin-activated receptor activity / positive regulation of toll-like receptor 2 signaling pathway / positive regulation of actin filament depolymerization / cell-cell junction maintenance / positive regulation of cytokine production involved in immune response / positive regulation of toll-like receptor 4 signaling pathway / T cell activation involved in immune response / positive regulation of pseudopodium assembly / negative regulation of chemokine production / positive regulation of phagocytosis, engulfment / positive regulation of chemotaxis / negative regulation of JNK cascade / neutrophil activation / establishment of endothelial barrier / regulation of canonical NF-kappaB signal transduction / positive regulation of positive chemotaxis / positive regulation of leukocyte chemotaxis / positive regulation of Rho protein signal transduction / leukocyte migration / pseudopodium / regulation of blood coagulation / positive regulation of interleukin-10 production / regulation of JNK cascade / negative regulation of tumor necrosis factor-mediated signaling pathway / G-protein alpha-subunit binding / negative regulation of insulin secretion / positive regulation of chemokine production / Peptide ligand-binding receptors / positive regulation of superoxide anion generation / positive regulation of GTPase activity / positive regulation of interleukin-1 beta production / G protein-coupled receptor activity / positive regulation of interleukin-8 production / electron transport chain / positive regulation of JNK cascade / vasodilation / positive regulation of interleukin-6 production / G-protein beta-subunit binding / positive regulation of type II interferon production / blood coagulation / signaling receptor activity / positive regulation of cytosolic calcium ion concentration / G alpha (q) signalling events / positive regulation of canonical NF-kappaB signal transduction / defense response to virus / protease binding / periplasmic space / electron transfer activity / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of ERK1 and ERK2 cascade / early endosome / positive regulation of cell migration / inflammatory response / iron ion binding / G protein-coupled receptor signaling pathway / signaling receptor binding / innate immune response / heme binding / Golgi apparatus / positive regulation of transcription by RNA polymerase II / plasma membrane
Similarity search - Function
Protease-activated receptor 2 / Protease-activated receptor / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family) / Immunoglobulins ...Protease-activated receptor 2 / Protease-activated receptor / Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / G protein-coupled receptor, rhodopsin-like / GPCR, rhodopsin-like, 7TM / G-protein coupled receptors family 1 profile. / 7 transmembrane receptor (rhodopsin family) / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
Soluble cytochrome b562 / Proteinase-activated receptor 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli (E. coli)
Mus musculus (house mouse)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 4 Å
AuthorsCheng, R.K.Y. / Fiez-Vandal, C. / Schlenker, O. / Edman, K. / Aggeler, B. / Brown, D.G. / Brown, G. / Cooke, R.M. / Dumelin, C.E. / Dore, A.S. ...Cheng, R.K.Y. / Fiez-Vandal, C. / Schlenker, O. / Edman, K. / Aggeler, B. / Brown, D.G. / Brown, G. / Cooke, R.M. / Dumelin, C.E. / Dore, A.S. / Geschwindner, S. / Grebner, C. / Hermansson, N.-O. / Jazayeri, A. / Johansson, P. / Leong, L. / Prihandoko, R. / Rappas, M. / Soutter, H. / Snijder, A. / Sundstrom, L. / Tehan, B. / Thornton, P. / Troast, D. / Wiggin, G. / Zhukov, A. / Marshall, F.H. / Dekker, N.
CitationJournal: Nature / Year: 2017
Title: Structural insight into allosteric modulation of protease-activated receptor 2.
Authors: Cheng, R.K.Y. / Fiez-Vandal, C. / Schlenker, O. / Edman, K. / Aggeler, B. / Brown, D.G. / Brown, G.A. / Cooke, R.M. / Dumelin, C.E. / Dore, A.S. / Geschwindner, S. / Grebner, C. / ...Authors: Cheng, R.K.Y. / Fiez-Vandal, C. / Schlenker, O. / Edman, K. / Aggeler, B. / Brown, D.G. / Brown, G.A. / Cooke, R.M. / Dumelin, C.E. / Dore, A.S. / Geschwindner, S. / Grebner, C. / Hermansson, N.O. / Jazayeri, A. / Johansson, P. / Leong, L. / Prihandoko, R. / Rappas, M. / Soutter, H. / Snijder, A. / Sundstrom, L. / Tehan, B. / Thornton, P. / Troast, D. / Wiggin, G. / Zhukov, A. / Marshall, F.H. / Dekker, N.
History
DepositionMar 28, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 3, 2017Provider: repository / Type: Initial release
Revision 1.1May 17, 2017Group: Database references
Revision 1.2Jan 17, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Proteinase-activated receptor 2,Soluble cytochrome b562,Proteinase-activated receptor 2
H: Fab3949 H
L: Fab3949 L


Theoretical massNumber of molelcules
Total (without water)96,1263
Polymers96,1263
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: gel filtration, 1:1 complex stoichiometry
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5660 Å2
ΔGint-35 kcal/mol
Surface area37340 Å2
MethodPISA
Unit cell
Length a, b, c (Å)164.144, 38.520, 159.660
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP21212

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Components

#1: Protein Proteinase-activated receptor 2,Soluble cytochrome b562,Proteinase-activated receptor 2 / PAR-2 / Coagulation factor II receptor-like 1 / G-protein coupled receptor 11 / Thrombin receptor- ...PAR-2 / Coagulation factor II receptor-like 1 / G-protein coupled receptor 11 / Thrombin receptor-like 1 / Cytochrome b-562 / PAR-2 / Coagulation factor II receptor-like 1 / G-protein coupled receptor 11 / Thrombin receptor-like 1


Mass: 49212.621 Da / Num. of mol.: 1
Mutation: G89A, H108A, G157A, M166L, Y174A, V176E, N222Q, M268A, I289A, L293A,G89A, H108A, G157A, M166L, Y174A, V176E, N222Q, M268A, I289A, L293A,G89A, H108A, G157A, M166L, Y174A, V176E, N222Q, M268A, I289A, L293A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Escherichia coli (E. coli)
Gene: F2RL1, GPR11, PAR2, cybC / Plasmid: pFASTBAC / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): Sf9 / References: UniProt: P55085, UniProt: P0ABE7
#2: Antibody Fab3949 H


Mass: 23590.354 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#3: Antibody Fab3949 L


Mass: 23322.725 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)

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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: 2.63 Å3/Da / Density % sol: 53.15 % / Description: NEEDLE SHAPED
Crystal growTemperature: 293 K / Method: lipidic cubic phase / pH: 5.7
Details: 0.1M MES pH 5.5-6.2, 0.2M POTASSIUM / SODIUM TARTRATE, 30-35% (W/V) PEG400, 2% (W/V) 2,5-HEXANEDIOL
PH range: 5.5-6.2

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Data collection

DiffractionMean temperature: 200 K
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID29 / Wavelength: 0.97717 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Sep 27, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97717 Å / Relative weight: 1
ReflectionResolution: 4→45.13 Å / Num. obs: 8440 / % possible obs: 93.6 % / Redundancy: 3.3 % / Biso Wilson estimate: 39.33 Å2 / CC1/2: 0.961 / Rmerge(I) obs: 0.315 / Rpim(I) all: 0.185 / Net I/σ(I): 4.2
Reflection shellResolution: 4→4.47 Å / Redundancy: 3.3 % / Rmerge(I) obs: 0.634 / Mean I/σ(I) obs: 2 / Num. unique obs: 2505 / CC1/2: 0.666 / Rpim(I) all: 0.401 / % possible all: 94.1

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Processing

Software
NameVersionClassification
BUSTER2.11.6refinement
XDS2014data reduction
Aimless0.3.11data scaling
PHASER2014phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5NDD

5ndd


Resolution: 4→45.13 Å / Cor.coef. Fo:Fc: 0.81 / Cor.coef. Fo:Fc free: 0.732 / Rfactor Rfree error: 0.01 / Cross valid method: THROUGHOUT / σ(F): 0 / SU Rfree Blow DPI: 1.149
RfactorNum. reflection% reflectionSelection details
Rfree0.319 395 4.69 %RANDOM
Rwork0.263 ---
obs0.265 8418 91.6 %-
Displacement parametersBiso mean: 71.15 Å2
Baniso -1Baniso -2Baniso -3
1--14.3757 Å20 Å20 Å2
2--8.7652 Å20 Å2
3---5.6105 Å2
Refine analyzeLuzzati coordinate error obs: 0.76 Å
Refinement stepCycle: 1 / Resolution: 4→45.13 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6467 0 0 0 6467
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0066626HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.89020HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d2198SINUSOIDAL2
X-RAY DIFFRACTIONt_incorr_chiral_ct
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_trig_c_planes124HARMONIC2
X-RAY DIFFRACTIONt_gen_planes961HARMONIC5
X-RAY DIFFRACTIONt_it6626HARMONIC20
X-RAY DIFFRACTIONt_nbd
X-RAY DIFFRACTIONt_omega_torsion1.83
X-RAY DIFFRACTIONt_other_torsion17.73
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_chiral_improper_torsion899SEMIHARMONIC5
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_contact7492SEMIHARMONIC4
LS refinement shellResolution: 4→4.47 Å / Rfactor Rfree error: 0 / Total num. of bins used: 5
RfactorNum. reflection% reflection
Rfree0.332 106 4.52 %
Rwork0.252 2238 -
all0.256 2344 -
obs--92.72 %

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