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- PDB-1b7x: STRUCTURE OF HUMAN ALPHA-THROMBIN Y225I MUTANT BOUND TO D-PHE-PRO... -

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Basic information

Entry
Database: PDB / ID: 1b7x
TitleSTRUCTURE OF HUMAN ALPHA-THROMBIN Y225I MUTANT BOUND TO D-PHE-PRO-ARG-CHLOROMETHYLKETONE
Components
  • PROTEIN (INHIBITOR)
  • PROTEIN (THROMBIN HEAVY CHAIN)
  • PROTEIN (THROMBIN LIGHT CHAIN)
KeywordsHYDROLASE/HYDROLASE INHIBITOR / SERINE PROTEASE / HYDROLASE-HYDROLASE INHIBITOR COMPLEX
Function / homology
Function and homology information


positive regulation of lipid kinase activity / positive regulation of phospholipase C-activating G protein-coupled receptor signaling pathway / cytolysis by host of symbiont cells / thrombospondin receptor activity / Defective factor XII causes hereditary angioedema / thrombin / neutrophil-mediated killing of gram-negative bacterium / regulation of blood coagulation / ligand-gated ion channel signaling pathway / Defective F8 cleavage by thrombin ...positive regulation of lipid kinase activity / positive regulation of phospholipase C-activating G protein-coupled receptor signaling pathway / cytolysis by host of symbiont cells / thrombospondin receptor activity / Defective factor XII causes hereditary angioedema / thrombin / neutrophil-mediated killing of gram-negative bacterium / regulation of blood coagulation / ligand-gated ion channel signaling pathway / Defective F8 cleavage by thrombin / Platelet Aggregation (Plug Formation) / negative regulation of platelet activation / negative regulation of astrocyte differentiation / positive regulation of collagen biosynthetic process / negative regulation of cytokine production involved in inflammatory response / positive regulation of blood coagulation / negative regulation of fibrinolysis / Gamma-carboxylation of protein precursors / Transport of gamma-carboxylated protein precursors from the endoplasmic reticulum to the Golgi apparatus / Common Pathway of Fibrin Clot Formation / Removal of aminoterminal propeptides from gamma-carboxylated proteins / fibrinolysis / regulation of cytosolic calcium ion concentration / Intrinsic Pathway of Fibrin Clot Formation / Peptide ligand-binding receptors / positive regulation of release of sequestered calcium ion into cytosol / Regulation of Complement cascade / acute-phase response / Cell surface interactions at the vascular wall / lipopolysaccharide binding / negative regulation of proteolysis / positive regulation of receptor signaling pathway via JAK-STAT / growth factor activity / positive regulation of insulin secretion / platelet activation / response to wounding / Golgi lumen / positive regulation of protein localization to nucleus / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / positive regulation of reactive oxygen species metabolic process / blood coagulation / antimicrobial humoral immune response mediated by antimicrobial peptide / Thrombin signalling through proteinase activated receptors (PARs) / heparin binding / regulation of cell shape / positive regulation of cell growth / G alpha (q) signalling events / collagen-containing extracellular matrix / blood microparticle / cell surface receptor signaling pathway / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of protein phosphorylation / G protein-coupled receptor signaling pathway / endoplasmic reticulum lumen / signaling receptor binding / serine-type endopeptidase activity / calcium ion binding / positive regulation of cell population proliferation / proteolysis / extracellular space / extracellular exosome / extracellular region / plasma membrane
Similarity search - Function
Prothrombin/thrombin / Thrombin light chain / Thrombin light chain domain superfamily / Thrombin light chain / Kringle domain / Kringle / Kringle, conserved site / Kringle superfamily / Kringle domain signature. / Kringle domain profile. ...Prothrombin/thrombin / Thrombin light chain / Thrombin light chain domain superfamily / Thrombin light chain / Kringle domain / Kringle / Kringle, conserved site / Kringle superfamily / Kringle domain signature. / Kringle domain profile. / Kringle domain / Vitamin K-dependent carboxylation/gamma-carboxyglutamic (GLA) domain / Gamma-carboxyglutamic acid-rich (GLA) domain / Gamma-carboxyglutamic acid-rich (GLA) domain superfamily / Vitamin K-dependent carboxylation domain. / Gla domain profile. / Domain containing Gla (gamma-carboxyglutamate) residues. / Kringle-like fold / Serine proteases, trypsin family, histidine active site / Serine proteases, trypsin family, serine active site / Peptidase S1A, chymotrypsin family / Serine proteases, trypsin family, histidine active site. / Serine proteases, trypsin domain profile. / Serine proteases, trypsin family, serine active site. / Trypsin-like serine protease / Serine proteases, trypsin domain / Trypsin / Trypsin-like serine proteases / Thrombin, subunit H / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / Beta Barrel / Mainly Beta
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.1 Å
AuthorsCaccia, S. / Futterer, K. / Di Cera, E. / Waksman, G.
CitationJournal: Proc.Natl.Acad.Sci.USA / Year: 1999
Title: Unexpected crucial role of residue 225 in serine proteases.
Authors: Guinto, E.R. / Caccia, S. / Rose, T. / Futterer, K. / Waksman, G. / Di Cera, E.
History
DepositionJan 25, 1999Deposition site: BNL / Processing site: RCSB
Revision 1.0Mar 2, 1999Provider: repository / Type: Initial release
Revision 1.1Apr 27, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Nov 3, 2021Group: Database references / Derived calculations / Category: database_2 / struct_conn / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_leaving_atom_flag / _struct_ref_seq_dif.details
Revision 1.4Aug 9, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: PROTEIN (THROMBIN LIGHT CHAIN)
B: PROTEIN (THROMBIN HEAVY CHAIN)
C: PROTEIN (INHIBITOR)


Theoretical massNumber of molelcules
Total (without water)34,2463
Polymers34,2463
Non-polymers00
Water2,630146
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3050 Å2
ΔGint-13 kcal/mol
Surface area11750 Å2
MethodPISA
Unit cell
Length a, b, c (Å)50.600, 73.700, 89.900
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein/peptide PROTEIN (THROMBIN LIGHT CHAIN)


Mass: 4096.534 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Cell line (production host): BABY HAMSTER KIDNEY CELLS (BHK-21)
Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P00734, thrombin
#2: Protein PROTEIN (THROMBIN HEAVY CHAIN)


Mass: 29730.203 Da / Num. of mol.: 1 / Mutation: Y225I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Cell line (production host): BABY HAMSTER KIDNEY CELLS (BHK-21)
Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P00734, thrombin
#3: Protein/peptide PROTEIN (INHIBITOR)


Mass: 419.498 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: D-PHE-PRO-ARG-CHLOROMETHYLKETONE / Source: (gene. exp.) Homo sapiens (human)
Cell line (production host): BABY HAMSTER KIDNEY CELLS (BHK-21)
Production host: Saccharomyces cerevisiae (brewer's yeast)
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 146 / Source method: isolated from a natural source / Formula: H2O
Nonpolymer detailsD-PHE-PRO-ARG-CHLOROMETHYLKETONE HAS FORMED TWO COVALENT CONNECTIONS TO THROMBIN: 1) VIA A ...D-PHE-PRO-ARG-CHLOROMETHYLKETONE HAS FORMED TWO COVALENT CONNECTIONS TO THROMBIN: 1) VIA A HEMIKETAL GROUP FROM C OF ARG C 303 TO OG OF SER B 195. 2) VIA A METHYLENE GROUP FROM CH2 OF ARG C 303 TO NE2 OF HIS B 57.

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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: 2.45 Å3/Da / Density % sol: 49.72 %
Crystal growpH: 7.5 / Details: pH 7.5
Crystal grow
*PLUS
Temperature: 20 ℃ / Method: vapor diffusion
Details: mutants are inhibited by a 10-fold molar excess of H-D-Phe-Pro-Arg-chloromethylketone for 30 min at room temperature
PH range low: 6.5 / PH range high: 6
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDChemical formula
13.3 mg/mlprotein1drop
25 mMMES1drop
3250 mM1dropNaCl
420 %PEG80001reservoir
50.1 Mzinc acetate1reservoir
60.1 Mcacodylate1reservoir

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 19-ID / Wavelength: 1.03
DetectorDate: Apr 15, 1998
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.03 Å / Relative weight: 1
ReflectionResolution: 2.1→30 Å / Num. obs: 16825 / % possible obs: 82.9 % / Observed criterion σ(I): 1 / Redundancy: 2.5 % / Biso Wilson estimate: 16.1 Å2 / Rmerge(I) obs: 0.049 / Rsym value: 0.049 / Net I/σ(I): 10
Reflection shellResolution: 2.1→2.18 Å / Redundancy: 2 % / Rmerge(I) obs: 0.104 / Mean I/σ(I) obs: 3.5 / Rsym value: 0.104 / % possible all: 81.9
Reflection
*PLUS
Lowest resolution: 30 Å / Num. measured all: 72954
Reflection shell
*PLUS
% possible obs: 81.9 %

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Processing

Software
NameVersionClassification
DENZOdata reduction
SCALEPACKdata scaling
AMoREphasing
X-PLOR3.851refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1PPB

1ppb


Resolution: 2.1→6 Å / Rfactor Rfree error: 0.01 / Data cutoff high absF: 10000000 / Data cutoff low absF: 0.001 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 2
RfactorNum. reflection% reflectionSelection details
Rfree0.282 804 4.9 %RANDOM
Rwork0.226 ---
obs0.226 16517 85.6 %-
Displacement parametersBiso mean: 19.1 Å2
Baniso -1Baniso -2Baniso -3
1-0 Å20 Å20 Å2
2--0 Å20 Å2
3---0 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.34 Å0.27 Å
Luzzati d res low-5 Å
Luzzati sigma a0.3 Å0.27 Å
Refinement stepCycle: LAST / Resolution: 2.1→6 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2115 0 0 146 2261
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONx_bond_d0.011
X-RAY DIFFRACTIONx_bond_d_na
X-RAY DIFFRACTIONx_bond_d_prot
X-RAY DIFFRACTIONx_angle_d
X-RAY DIFFRACTIONx_angle_d_na
X-RAY DIFFRACTIONx_angle_d_prot
X-RAY DIFFRACTIONx_angle_deg1.6
X-RAY DIFFRACTIONx_angle_deg_na
X-RAY DIFFRACTIONx_angle_deg_prot
X-RAY DIFFRACTIONx_dihedral_angle_d27.8
X-RAY DIFFRACTIONx_dihedral_angle_d_na
X-RAY DIFFRACTIONx_dihedral_angle_d_prot
X-RAY DIFFRACTIONx_improper_angle_d0.86
X-RAY DIFFRACTIONx_improper_angle_d_na
X-RAY DIFFRACTIONx_improper_angle_d_prot
X-RAY DIFFRACTIONx_mcbond_it2.041.5
X-RAY DIFFRACTIONx_mcangle_it2.992
X-RAY DIFFRACTIONx_scbond_it3.082
X-RAY DIFFRACTIONx_scangle_it4.332.5
LS refinement shellResolution: 2.1→2.19 Å / Rfactor Rfree error: 0.032 / Total num. of bins used: 8
RfactorNum. reflection% reflection
Rfree0.334 109 5.3 %
Rwork0.294 1949 -
obs--85.9 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMTOPHCSDX_CH2.PRO
X-RAY DIFFRACTION2ION.PARAM
Refinement
*PLUS
Highest resolution: 2.1 Å / Lowest resolution: 6 Å / % reflection Rfree: 4 % / Rfactor Rwork: 0.225
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONx_angle_deg1.58
X-RAY DIFFRACTIONx_dihedral_angle_d
X-RAY DIFFRACTIONx_dihedral_angle_deg27.8
X-RAY DIFFRACTIONx_improper_angle_d
X-RAY DIFFRACTIONx_improper_angle_deg0.86

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