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- PDB-1oyt: COMPLEX OF RECOMBINANT HUMAN THROMBIN WITH A DESIGNED FLUORINATED... -

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Basic information

Entry
Database: PDB / ID: 1oyt
TitleCOMPLEX OF RECOMBINANT HUMAN THROMBIN WITH A DESIGNED FLUORINATED INHIBITOR
Components
  • Hirudin IIB
  • thrombin heavy chain
  • thrombin light chain
KeywordsHYDROLASE/HYDROLASE INHIBITOR / SERINE PROTEINASE / 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 / Transport of gamma-carboxylated protein precursors from the endoplasmic reticulum to the Golgi apparatus / regulation of cytosolic calcium ion concentration / 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 / serine-type endopeptidase inhibitor activity / 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
Hirudin / Proteinase inhibitor I14, hirudin / Thrombin inhibitor hirudin / Hirudin/antistatin / Prothrombin/thrombin / Thrombin light chain / Thrombin light chain domain superfamily / : / Thrombin light chain / Kringle domain ...Hirudin / Proteinase inhibitor I14, hirudin / Thrombin inhibitor hirudin / Hirudin/antistatin / 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
N-terminal Asp des-amino Hirudin-3A / Chem-FSN / Prothrombin / Hirudin-2B
Similarity search - Component
Biological speciesHomo sapiens (human)
Hirudo medicinalis (medicinal leech)
MethodX-RAY DIFFRACTION / FOURIER SYNTHESIS / Resolution: 1.67 Å
AuthorsBanner, D.W. / Olsen, J.A.
Citation
Journal: Angew.Chem.Int.Ed.Engl. / Year: 2003
Title: A Fluorine Scan of Thrombin Inhibitors to Map the Fluorophilicity/Fluorophobicity of an Enzyme Active Site: Evidence for CF...C=O Interactions.
Authors: Olsen, J.A. / Banner, D.W. / Seiler, P. / Obst-Sander, U. / D'Arcy, A. / Stihle, M. / Mueller, K. / Diederich, F.
#1: Journal: Protein Expr.Purif. / Year: 1997
Title: Stable expression and purification of a secreted human recombinant prethrombin-2 and its activation to thrombin
Authors: Russo, G. / Gast, A. / Schlaeger, E.J. / Angiolillo, A. / Pietropaolo, C.
History
DepositionApr 7, 2003Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 24, 2003Provider: repository / Type: Initial release
Revision 1.1Apr 29, 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 1.3Dec 12, 2012Group: Other
Revision 1.4May 25, 2016Group: Source and taxonomy
Revision 1.5Oct 11, 2017Group: Advisory / Refinement description / Category: pdbx_unobs_or_zero_occ_atoms / software / Item: _software.classification / _software.name
Revision 1.6Apr 3, 2024Group: Advisory / Data collection ...Advisory / 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 / pdbx_unobs_or_zero_occ_atoms / struct_conn / struct_ref_seq_dif / 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_PDB_ins_code / _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_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr1_symmetry / _pdbx_struct_conn_angle.ptnr2_auth_comp_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr2_label_atom_id / _pdbx_struct_conn_angle.ptnr2_label_comp_id / _pdbx_struct_conn_angle.ptnr3_PDB_ins_code / _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_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.ptnr3_symmetry / _pdbx_struct_conn_angle.value / _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.ptnr1_symmetry / _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.ptnr2_symmetry / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
L: thrombin light chain
H: thrombin heavy chain
I: Hirudin IIB
hetero molecules


Theoretical massNumber of molelcules
Total (without water)35,7446
Polymers35,2733
Non-polymers4713
Water7,278404
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4270 Å2
ΔGint-35 kcal/mol
Surface area12690 Å2
MethodPISA
Unit cell
Length a, b, c (Å)70.693, 71.479, 72.802
Angle α, β, γ (deg.)90.00, 100.53, 90.00
Int Tables number5
Space group name H-MC121
Components on special symmetry positions
IDModelComponents
11H-780-

HOH

21I-107-

HOH

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Components

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Protein/peptide , 2 types, 2 molecules LI

#1: Protein/peptide thrombin light chain / Coagulation factor II


Mass: 4096.534 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F2 / Cell (production host): OVARY / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P00734, thrombin
#3: Protein/peptide Hirudin IIB


Type: Oligopeptide / Class: Anticoagulant, Antithrombotic / Mass: 1396.450 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: The sequence was chemically synthesized. The sequence is naturally found in Hirudo medicinalis (Medicinal leech).
Source: (synth.) Hirudo medicinalis (medicinal leech)
References: UniProt: P28506, N-terminal Asp des-amino Hirudin-3A

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Protein , 1 types, 1 molecules H

#2: Protein thrombin heavy chain / Coagulation factor II


Mass: 29780.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F2 / Cell (production host): OVARY / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P00734, thrombin

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Non-polymers , 4 types, 407 molecules

#4: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#5: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#6: Chemical ChemComp-FSN / (3ASR,4RS,8ASR,8BRS)-4-(2-(4-FLUOROBENZYL)-1,3-DIOXODEACAHYDROPYRROLO[3,4-A] PYRROLIZIN-4-YL)BENZAMIDINE


Mass: 407.461 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C23H24FN4O2
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 404 / 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: 2.56 Å3/Da / Density % sol: 51.99 %
Crystal grow
*PLUS
pH: 7.4 / Method: unknown
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDChemical formulaDetails
130 mMPEG335011
2100 mM11NaCl
3100 mMsodium potassium phosphate11pH7.4

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: ENRAF-NONIUS FR591 / Wavelength: 1.5418 Å
DetectorType: MAR scanner 345 mm plate / Detector: IMAGE PLATE / Date: May 24, 2002 / Details: Osmic mirrors
RadiationMonochromator: OSMICS / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.64→20 Å / Num. all: 44106 / Num. obs: 38758 / % possible obs: 87.9 % / Observed criterion σ(I): -3 / Redundancy: 7.33 % / Biso Wilson estimate: 21.8 Å2 / Rmerge(I) obs: 0.036 / Rsym value: 0.042 / Net I/σ(I): 33.43
Reflection shellResolution: 1.64→1.74 Å / Redundancy: 6.25 % / Rmerge(I) obs: 0.16 / Mean I/σ(I) obs: 9.71 / Num. unique all: 7048 / Rsym value: 0.17 / % possible all: 53
Reflection
*PLUS
Highest resolution: 1.67 Å / Num. measured all: 284109 / Rmerge(I) obs: 0.033
Reflection shell
*PLUS
Rmerge(I) obs: 0.149 / Mean I/σ(I) obs: 9.7

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Processing

Software
NameVersionClassification
CNX2000refinement
MAR345data collection
XDSdata scaling
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: IN HOUSE THROMBIN STRUCTURES

Resolution: 1.67→19.3 Å / Rfactor Rfree error: 0.005 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh & Huber
Details: CHYMOTRYPSIN NUMBERING (RATHER THAN SEQUENTIAL) SYSTEM IS USED, BASED ON THE TOPOLOGICAL ALIGNMENT WITH THE STRUCTURE OF CHYMOTRYPSIN (W.BODE ET AL., 1989, EMBO J. 8, 3467-3475). Double ...Details: CHYMOTRYPSIN NUMBERING (RATHER THAN SEQUENTIAL) SYSTEM IS USED, BASED ON THE TOPOLOGICAL ALIGNMENT WITH THE STRUCTURE OF CHYMOTRYPSIN (W.BODE ET AL., 1989, EMBO J. 8, 3467-3475). Double confromations given as single conformer are: H chain Leu41, Arg50, Leu64, Val66, Leu85, Met106, Ser129B Leu130, Asp186. Gly186C has a second trace with psi of 186D + 180degrees. Missing residues H chain: 148 loop WTANVGK, residues Thr147 and Gln151 weak. Missing residues L chain: N-terminus TFGSGE, C-terminus DGR. Missing from 'hirugen' I chain: C-terminus EEYLQ. Hirugen corresponds to hirudin 55-65, is synthesized chemically, is not sulphated. Asp 55 has no amino group and thus is given as a succinic acid residue. Arg75 has 2 conformations. COORDINATES ARE GIVEN UP TO CB. WATERS 107 AND 35 INDICATE THE GUANIDINIUM POSITION ON THE 2-FOLD AXIS. Residues with some or all atoms at 0.5 occupancy are L chain: Asp1A, Lys14A, Arg14D, Ile14K, H chain: Leu41, Lys81, Lys110, Pro111, Arg126, Glu127, Ser129B, Thr147, Gln151, Asp186A, Lys186D, Glu192, Asn205, Lys236 Gln239, Lys240, Gln244, I chain Glu4, Pro6. Atoms with zero occupancy H chain Ser36A Og and Asn62 OD1. The sugar on Asn62 is not modelled (water molecules). All inhibitor atoms refine to B-values < 25. The weakest is O18. Between O18 and the NZ of K60F is a smear of density, not well fitted by water 233.
RfactorNum. reflection% reflectionSelection details
Rfree0.205 1938 5 %RANDOM BUT NOT SAME AS FOR PREVIOUS REFINED STRUCTURES
Rwork0.179 ---
obs-38750 93.6 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 48.7734 Å2 / ksol: 0.312145 e/Å3
Displacement parametersBiso mean: 17.5 Å2
Baniso -1Baniso -2Baniso -3
1-0.95 Å20 Å2-0.76 Å2
2---1.35 Å20 Å2
3---0.41 Å2
Refine analyzeLuzzati coordinate error free: 0.2 Å / Luzzati sigma a free: 0.12 Å
Refinement stepCycle: LAST / Resolution: 1.67→19.3 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2292 0 32 404 2728
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.011
X-RAY DIFFRACTIONc_angle_deg1.8
X-RAY DIFFRACTIONc_dihedral_angle_d24.3
X-RAY DIFFRACTIONc_improper_angle_d2.06
X-RAY DIFFRACTIONc_mcbond_it0.631.5
X-RAY DIFFRACTIONc_mcangle_it1.092
X-RAY DIFFRACTIONc_scbond_it1.472
X-RAY DIFFRACTIONc_scangle_it2.142.5
LS refinement shellResolution: 1.67→1.73 Å / Rfactor Rfree error: 0.019 / Total num. of bins used: 10
RfactorNum. reflection% reflection
Rfree0.249 181 5 %
Rwork0.228 3436 -
obs--87.9 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2ROCHE.PARAMROCHE.TOP
X-RAY DIFFRACTION3FSN.PRXFSN.TPX
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg24.3
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg2.06

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