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- PDB-4yz9: Crystal Structure of human phosphorylated IRE1alpha in complex wi... -

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Basic information

Entry
Database: PDB / ID: 4yz9
TitleCrystal Structure of human phosphorylated IRE1alpha in complex with a type III kinase inhibitor (GSK2850163A)
ComponentsSerine/threonine-protein kinase/endoribonuclease IRE1
KeywordsTRANSFERASE/TRANSFERASE INHIBITOR / active / inhibitor / complex / IRE1 / TRANSFERASE-TRANSFERASE INHIBITOR complex
Function / homology
Function and homology information


peptidyl-serine trans-autophosphorylation / mRNA splicing, via endonucleolytic cleavage and ligation / AIP1-IRE1 complex / Ire1 complex / IRE1alpha activates chaperones / IRE1-TRAF2-ASK1 complex / insulin metabolic process / positive regulation of endoplasmic reticulum unfolded protein response / IRE1-RACK1-PP2A complex / platelet-derived growth factor receptor binding ...peptidyl-serine trans-autophosphorylation / mRNA splicing, via endonucleolytic cleavage and ligation / AIP1-IRE1 complex / Ire1 complex / IRE1alpha activates chaperones / IRE1-TRAF2-ASK1 complex / insulin metabolic process / positive regulation of endoplasmic reticulum unfolded protein response / IRE1-RACK1-PP2A complex / platelet-derived growth factor receptor binding / nuclear inner membrane / endothelial cell proliferation / Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters / IRE1-mediated unfolded protein response / mRNA catabolic process / positive regulation of JUN kinase activity / intrinsic apoptotic signaling pathway in response to endoplasmic reticulum stress / cellular response to vascular endothelial growth factor stimulus / cellular response to unfolded protein / regulation of macroautophagy / positive regulation of vascular associated smooth muscle cell proliferation / Hsp70 protein binding / RNA endonuclease activity / response to endoplasmic reticulum stress / positive regulation of RNA splicing / cellular response to glucose stimulus / Hsp90 protein binding / ADP binding / cellular response to hydrogen peroxide / unfolded protein binding / protein phosphorylation / non-specific serine/threonine protein kinase / protein serine kinase activity / protein serine/threonine kinase activity / endoplasmic reticulum membrane / enzyme binding / magnesium ion binding / endoplasmic reticulum / protein homodimerization activity / mitochondrion / ATP binding / identical protein binding / cytoplasm
Similarity search - Function
KEN domain / Serine/threonine-protein kinase/endoribonuclease IRE1/2-like / KEN domain / KEN domain superfamily / Ribonuclease 2-5A / KEN domain profile. / domain in protein kinases, N-glycanases and other nuclear proteins / de novo design (two linked rop proteins) / Pyrrolo-quinoline quinone beta-propeller repeat / beta-propeller repeat ...KEN domain / Serine/threonine-protein kinase/endoribonuclease IRE1/2-like / KEN domain / KEN domain superfamily / Ribonuclease 2-5A / KEN domain profile. / domain in protein kinases, N-glycanases and other nuclear proteins / de novo design (two linked rop proteins) / Pyrrolo-quinoline quinone beta-propeller repeat / beta-propeller repeat / Quinoprotein alcohol dehydrogenase-like superfamily / Phosphorylase Kinase; domain 1 / Phosphorylase Kinase; domain 1 / Transferase(Phosphotransferase) domain 1 / Transferase(Phosphotransferase); domain 1 / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / Up-down Bundle / 2-Layer Sandwich / Orthogonal Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Chem-4K7 / Serine/threonine-protein kinase/endoribonuclease IRE1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 2.463 Å
AuthorsConcha, N.O.
CitationJournal: Mol.Pharmacol. / Year: 2015
Title: Long-Range Inhibitor-Induced Conformational Regulation of Human IRE1 alpha Endoribonuclease Activity.
Authors: Concha, N.O. / Smallwood, A. / Bonnette, W. / Totoritis, R. / Zhang, G. / Federowicz, K. / Yang, J. / Qi, H. / Chen, S. / Campobasso, N. / Choudhry, A.E. / Shuster, L.E. / Evans, K.A. / ...Authors: Concha, N.O. / Smallwood, A. / Bonnette, W. / Totoritis, R. / Zhang, G. / Federowicz, K. / Yang, J. / Qi, H. / Chen, S. / Campobasso, N. / Choudhry, A.E. / Shuster, L.E. / Evans, K.A. / Ralph, J. / Sweitzer, S. / Heerding, D.A. / Buser, C.A. / Su, D.S. / DeYoung, M.P.
History
DepositionMar 24, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 11, 2015Provider: repository / Type: Initial release
Revision 1.1Sep 27, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2 / pdbx_initial_refinement_model / pdbx_struct_oper_list
Item: _citation.journal_id_CSD / _database_2.pdbx_DOI ..._citation.journal_id_CSD / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.symmetry_operation

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Serine/threonine-protein kinase/endoribonuclease IRE1
B: Serine/threonine-protein kinase/endoribonuclease IRE1
C: Serine/threonine-protein kinase/endoribonuclease IRE1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)141,1716
Polymers139,8323
Non-polymers1,3393
Water1,44180
1
A: Serine/threonine-protein kinase/endoribonuclease IRE1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)47,0572
Polymers46,6111
Non-polymers4461
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Serine/threonine-protein kinase/endoribonuclease IRE1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)47,0572
Polymers46,6111
Non-polymers4461
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: Serine/threonine-protein kinase/endoribonuclease IRE1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)47,0572
Polymers46,6111
Non-polymers4461
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)244.004, 77.767, 88.313
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 Serine/threonine-protein kinase/endoribonuclease IRE1 / Endoplasmic reticulum-to-nucleus signaling 1 / Inositol-requiring protein 1 / hIRE1p / Ire1-alpha / IRE1a


Mass: 46610.570 Da / Num. of mol.: 3 / Fragment: UNP residues 562-966
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ERN1, IRE1 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: O75460, non-specific serine/threonine protein kinase, Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters
#2: Chemical ChemComp-4K7 / (5R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)-2,7-diazaspiro[4.5]decane-7-carboxamide


Mass: 446.413 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C24H29Cl2N3O
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 80 / 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: 3.01 Å3/Da / Density % sol: 59.12 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7
Details: Crystals of pIRE1alpha (547 - 977) with GSK2850163 were prepared by mixing pIRE1alpha with 0.5 mM GSK2850163 and incubating overnight on ice. The crystals were grown at 20 C by vapor ...Details: Crystals of pIRE1alpha (547 - 977) with GSK2850163 were prepared by mixing pIRE1alpha with 0.5 mM GSK2850163 and incubating overnight on ice. The crystals were grown at 20 C by vapor diffusion in sitting drops containing 2 ul of protein (13 mg/ml in 50 mM Hepes, pH 7.5, 200 mM NaCl, 5 mM DTT, 1 mM EDTA , 0.5 mM GSK2850163, 0.25% DMSO ) and 2 ul reservoir solution containing PEG 3350 (16%-22%), 100 mM Hepes pH 7.0, 200 mM Ca2+ acetate. The crystals were thick rods that appeared over 2-5 days and reached full size (0.05 X 0.025 X 0.3 mm) in 2 weeks. Seeding was used to improve crystal quality. The pIRE1alpha + GSK2850163 crystals were frozen in a solution of 20% ethylene glycol, 22% PEG 3350, 0.2 M calcium acetate added in a stepwise manner to the protein drop before mounting the crystal on the loop.

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Data collection

DiffractionMean temperature: 100 K / Ambient temp details: from a single crystal
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 / Wavelength: 1.54 Å
DetectorType: RAYONIX SX-165mm / Detector: CCD / Date: Oct 4, 2011
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54 Å / Relative weight: 1
ReflectionResolution: 2.45→29.182 Å / Num. obs: 59737 / % possible obs: 97.2 % / Observed criterion σ(I): 2 / Redundancy: 6.3 % / Biso Wilson estimate: 44.87 Å2 / Rmerge(I) obs: 0.073 / Rsym value: 0.073 / Χ2: 1.009 / Net I/av σ(I): 22.874 / Net I/σ(I): 9 / Num. measured all: 377173
Reflection shell

Diffraction-ID: 1 / Rejects: _

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique allΧ2% possible all
2.45-2.546.30.5923.154110.97289.7
2.54-2.646.20.4556520.99293.1
2.64-2.766.10.34858231.04395.8
2.76-2.96.10.26858781.02797
2.9-3.096.10.1860081.01998.5
3.09-3.326.20.11660281.01199.1
3.32-3.666.30.07661371.01299.4
3.66-4.196.40.06261331.01299.6
4.19-5.276.80.0436199199.8
5.27-306.60.0286468199.5

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Processing

Software
NameVersionClassification
PHENIX1.9_1692refinement
SCALEPACKdata scaling
PDB_EXTRACT3.15data extraction
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3P23

3p23


Resolution: 2.463→29.182 Å / SU ML: 0.35 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 33.01 / Stereochemistry target values: MLHL
RfactorNum. reflection% reflectionSelection details
Rfree0.2829 3016 5.06 %rqandom
Rwork0.2354 ---
obs0.2378 59654 96.69 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.463→29.182 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8931 0 90 80 9101
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0039280
X-RAY DIFFRACTIONf_angle_d0.66312591
X-RAY DIFFRACTIONf_dihedral_angle_d14.0453386
X-RAY DIFFRACTIONf_chiral_restr0.0381375
X-RAY DIFFRACTIONf_plane_restr0.0051624
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.4633-2.50180.3831080.31892095X-RAY DIFFRACTION79
2.5018-2.54280.34171300.30372319X-RAY DIFFRACTION89
2.5428-2.58660.33151310.30312432X-RAY DIFFRACTION92
2.5866-2.63360.39361250.2952416X-RAY DIFFRACTION93
2.6336-2.68430.37111320.29322552X-RAY DIFFRACTION95
2.6843-2.7390.32351370.28882477X-RAY DIFFRACTION96
2.739-2.79850.34411350.29012527X-RAY DIFFRACTION96
2.7985-2.86360.32171400.3092567X-RAY DIFFRACTION98
2.8636-2.93510.34471400.28532589X-RAY DIFFRACTION98
2.9351-3.01440.30741410.28612571X-RAY DIFFRACTION98
3.0144-3.1030.32381290.27562620X-RAY DIFFRACTION99
3.103-3.2030.31161470.2822609X-RAY DIFFRACTION99
3.203-3.31740.29471120.27462634X-RAY DIFFRACTION99
3.3174-3.450.36141580.27082636X-RAY DIFFRACTION99
3.45-3.60670.30591390.25482637X-RAY DIFFRACTION99
3.6067-3.79650.31221600.23362648X-RAY DIFFRACTION100
3.7965-4.03380.29241370.21152667X-RAY DIFFRACTION100
4.0338-4.34430.21251440.20412657X-RAY DIFFRACTION100
4.3443-4.77980.24241350.18432684X-RAY DIFFRACTION100
4.7798-5.46750.23161420.19562719X-RAY DIFFRACTION100
5.4675-6.87360.26061410.21792756X-RAY DIFFRACTION100
6.8736-29.1840.21731530.18492826X-RAY DIFFRACTION98

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