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- PDB-2thf: STRUCTURE OF HUMAN ALPHA-THROMBIN Y225F MUTANT BOUND TO D-PHE-PRO... -

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Basic information

Entry
Database: PDB / ID: 2thf
TitleSTRUCTURE OF HUMAN ALPHA-THROMBIN Y225F MUTANT BOUND TO D-PHE-PRO-ARG-CHLOROMETHYLKETONE
Components
  • THROMBIN HEAVY CHAIN
  • THROMBIN LIGHT CHAIN
KeywordsHYDROLASE/HYDROLASE INHIBITOR / SERINE PROTEASE / HYDROLASE-HYDROLASE INHIBITOR complex
Function / homology
Function and homology information


cytolysis by host of symbiont cells / thrombospondin receptor activity / Defective factor XII causes hereditary angioedema / thrombin / thrombin-activated receptor signaling pathway / negative regulation of astrocyte differentiation / regulation of blood coagulation / positive regulation of phospholipase C-activating G protein-coupled receptor signaling pathway / neutrophil-mediated killing of gram-negative bacterium / Defective F8 cleavage by thrombin ...cytolysis by host of symbiont cells / thrombospondin receptor activity / Defective factor XII causes hereditary angioedema / thrombin / thrombin-activated receptor signaling pathway / negative regulation of astrocyte differentiation / regulation of blood coagulation / positive regulation of phospholipase C-activating G protein-coupled receptor signaling pathway / neutrophil-mediated killing of gram-negative bacterium / Defective F8 cleavage by thrombin / Platelet Aggregation (Plug Formation) / ligand-gated ion channel signaling pathway / positive regulation of collagen biosynthetic process / negative regulation of platelet activation / negative regulation of blood coagulation / positive regulation of blood coagulation / negative regulation of fibrinolysis / regulation of cytosolic calcium ion concentration / Transport of gamma-carboxylated protein precursors from the endoplasmic reticulum to the Golgi apparatus / Gamma-carboxylation of protein precursors / Common Pathway of Fibrin Clot Formation / Removal of aminoterminal propeptides from gamma-carboxylated proteins / fibrinolysis / Intrinsic Pathway of Fibrin Clot Formation / negative regulation of proteolysis / negative regulation of cytokine production involved in inflammatory response / Peptide ligand-binding receptors / Regulation of Complement cascade / positive regulation of release of sequestered calcium ion into cytosol / acute-phase response / Cell surface interactions at the vascular wall / positive regulation of receptor signaling pathway via JAK-STAT / growth factor activity / lipopolysaccharide binding / positive regulation of insulin secretion / platelet activation / response to wounding / positive regulation of protein localization to nucleus / Golgi lumen / 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 / regulation of cell shape / heparin binding / Thrombin signalling through proteinase activated receptors (PARs) / : / positive regulation of cell growth / blood microparticle / G alpha (q) signalling events / cell surface receptor signaling pathway / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / receptor ligand activity / endoplasmic reticulum lumen / signaling receptor binding / serine-type endopeptidase activity / positive regulation of cell population proliferation / calcium ion binding / 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. ...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 / Serine proteases, trypsin family, histidine active site. / Peptidase S1A, chymotrypsin family / Serine proteases, trypsin family, serine active site. / Serine proteases, trypsin domain profile. / 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
D-Phe-Pro-Arg-CH2Cl / Chem-0G6 / Prothrombin
Similarity search - Component
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 26, 1999Deposition site: BNL / Processing site: RCSB
Revision 1.0Mar 7, 1999Provider: repository / Type: Initial release
Revision 1.1Apr 26, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Atomic model / Database references ...Atomic model / Database references / Derived calculations / Non-polymer description / Structure summary / Version format compliance
Revision 1.3Feb 27, 2013Group: Other
Revision 1.4Aug 30, 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 / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_conn_type / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.pdbx_ptnr1_PDB_ins_code / _struct_conn.pdbx_ptnr2_PDB_ins_code / _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_conn.ptnr2_label_seq_id / _struct_conn_type.id / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.5Nov 20, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: THROMBIN LIGHT CHAIN
B: THROMBIN HEAVY CHAIN
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,3384
Polymers33,8612
Non-polymers4772
Water2,522140
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3300 Å2
ΔGint-25 kcal/mol
Surface area11880 Å2
MethodPISA
Unit cell
Length a, b, c (Å)53.100, 75.100, 81.400
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 THROMBIN LIGHT CHAIN


Mass: 4096.534 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P00734
#2: Protein THROMBIN HEAVY CHAIN


Mass: 29764.219 Da / Num. of mol.: 1 / Mutation: Y225F
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: Chemical ChemComp-0G6 / D-phenylalanyl-N-[(2S,3S)-6-{[amino(iminio)methyl]amino}-1-chloro-2-hydroxyhexan-3-yl]-L-prolinamide / PPACK


Type: peptide-like, Peptide-like / Class: Inhibitor / Mass: 453.986 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H34ClN6O3 / Details: D-PHE-PRO-ARG-CHLOROMETHYLKETONE / References: D-Phe-Pro-Arg-CH2Cl
#4: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 140 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY
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 TO OG SER B 195. 2) VIA A METHYLENE GROUP TO NE2 HIS B 57.
Sequence detailsTHE PROTEIN USED IN THE PRESENT STRUCTURE DETERMINATION COMPRISED AMINO ACIDS 1H TO 247 ...THE PROTEIN USED IN THE PRESENT STRUCTURE DETERMINATION COMPRISED AMINO ACIDS 1H TO 247 (CHYMOTRYPSIN NUMBERING) OF HUMAN ALPHA THROMBIN. RESIDUE TYR B 225 WAS MUTATED TO PHE. DUE TO LACK OF ELECTRON DENSITY RESIDUES 1H THROUGH 1B (7 TOTAL) COULD NOT BE BUILT, AS WELL AS RESIDUES 147 THROUGH 149E (8 TOTAL), AND RESIDUES 246 AND 247.

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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: 2.37 Å3/Da / Density % sol: 48.02 %
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 1, 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: 18509 / % possible obs: 94.1 % / Observed criterion σ(I): 1 / Redundancy: 2.5 % / Biso Wilson estimate: 18.5 Å2 / Rmerge(I) obs: 0.048 / Rsym value: 0.048 / Net I/σ(I): 10
Reflection shellResolution: 2.1→2.18 Å / Redundancy: 2 % / Rmerge(I) obs: 0.131 / Mean I/σ(I) obs: 3.5 / Rsym value: 0.131 / % possible all: 89.6
Reflection
*PLUS
Num. measured all: 68774
Reflection shell
*PLUS
% possible obs: 89.6 %

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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: 0.007 / 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
Rfree0.265 1367 7.6 %RANDOM
Rwork0.199 ---
obs-18085 96.9 %-
Displacement parametersBiso mean: 22.7 Å2
Baniso -1Baniso -2Baniso -3
1-0 Å20 Å20 Å2
2--0 Å20 Å2
3---0 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.31 Å0.23 Å
Luzzati d res low-5 Å
Luzzati sigma a0.22 Å0.21 Å
Refinement stepCycle: LAST / Resolution: 2.1→6 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2207 0 31 140 2378
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONx_bond_d0.01
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_d28.4
X-RAY DIFFRACTIONx_dihedral_angle_d_na
X-RAY DIFFRACTIONx_dihedral_angle_d_prot
X-RAY DIFFRACTIONx_improper_angle_d0.78
X-RAY DIFFRACTIONx_improper_angle_d_na
X-RAY DIFFRACTIONx_improper_angle_d_prot
X-RAY DIFFRACTIONx_mcbond_it1.851.5
X-RAY DIFFRACTIONx_mcangle_it2.82
X-RAY DIFFRACTIONx_scbond_it3.212
X-RAY DIFFRACTIONx_scangle_it4.622.5
LS refinement shellResolution: 2.1→2.19 Å / Rfactor Rfree error: 0.023 / Total num. of bins used: 8
RfactorNum. reflection% reflection
Rfree0.288 157 7.2 %
Rwork0.24 2028 -
obs--95 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMTOPHCSDX_CH2.PRO
X-RAY DIFFRACTION2MTH.PARTOPHCSDX_CH2.PRO
X-RAY DIFFRACTION3ION.PARAMTOPHCSDX_CH2.PRO
X-RAY DIFFRACTION4MTH.PARTOPHCSDX_CH2.PRO
Software
*PLUS
Name: X-PLOR / Version: 3.851 / Classification: refinement
Refinement
*PLUS
Rfactor obs: 0.199
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONx_dihedral_angle_d
X-RAY DIFFRACTIONx_dihedral_angle_deg28.4
X-RAY DIFFRACTIONx_improper_angle_d
X-RAY DIFFRACTIONx_improper_angle_deg0.78
LS refinement shell
*PLUS
Rfactor obs: 0.24

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