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- PDB-4h6s: Crystal structure of thrombin mutant E14eA/D14lA/E18A/S195A -

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Basic information

Entry
Database: PDB / ID: 4h6s
TitleCrystal structure of thrombin mutant E14eA/D14lA/E18A/S195A
Components(ProthrombinThrombin) x 2
KeywordsHYDROLASE/PEPTIDE / Serine protease / prethrombin-2 / autoactivation / hydrolase / HYDROLASE-PEPTIDE 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 / regulation of blood coagulation / ligand-gated ion channel signaling pathway / neutrophil-mediated killing of gram-negative bacterium / 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 / regulation of blood coagulation / ligand-gated ion channel signaling pathway / neutrophil-mediated killing of gram-negative bacterium / Defective F8 cleavage by thrombin / Platelet Aggregation (Plug Formation) / negative regulation of astrocyte differentiation / negative regulation of platelet activation / 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 / positive regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / cell surface receptor signaling pathway / 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 / MOLECULAR REPLACEMENT / Resolution: 2.19 Å
AuthorsPozzi, N. / Chen, Z. / Zapata, F. / Pelc, L.A. / Di Cera, E.
Citation
Journal: J.Biol.Chem. / Year: 2013
Title: Autoactivation of thrombin precursors.
Authors: Pozzi, N. / Chen, Z. / Zapata, F. / Niu, W. / Barranco-Medina, S. / Pelc, L.A. / Di Cera, E.
#1: Journal: J.Biol.Chem. / Year: 2004
Title: Molecular dissection of Na+ binding to thrombin.
Authors: Pineda, A.O. / Carrell, C.J. / Bush, L.A. / Prasad, S. / Caccia, S. / Chen, Z.W. / Mathews, F.S. / Di Cera, E.
History
DepositionSep 19, 2012Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 13, 2013Provider: repository / Type: Initial release
Revision 1.1Jun 11, 2014Group: Database references
Revision 1.2Sep 20, 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_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.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.value / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_ptnr1_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_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
A: Prothrombin
B: Prothrombin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)33,2743
Polymers33,2512
Non-polymers231
Water1,856103
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2350 Å2
ΔGint-24 kcal/mol
Surface area13190 Å2
MethodPISA
Unit cell
Length a, b, c (Å)63.904, 70.828, 56.103
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/peptide Prothrombin / Thrombin / Coagulation factor II / Activation peptide fragment 1 / Activation peptide fragment 2 / Thrombin ...Coagulation factor II / Activation peptide fragment 1 / Activation peptide fragment 2 / Thrombin light chain / Thrombin heavy chain


Mass: 3545.029 Da / Num. of mol.: 1 / Mutation: E14eA,D14lA,E18A,S195A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F2 / Production host: Escherichia coli (E. coli) / References: UniProt: P00734, thrombin
#2: Protein Prothrombin / Thrombin / Coagulation factor II / Activation peptide fragment 1 / Activation peptide fragment 2 / Thrombin ...Coagulation factor II / Activation peptide fragment 1 / Activation peptide fragment 2 / Thrombin light chain / Thrombin heavy chain


Mass: 29706.184 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F2 / Production host: Escherichia coli (E. coli) / References: UniProt: P00734, thrombin
#3: Chemical ChemComp-NA / SODIUM ION


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


Mass: 18.015 Da / Num. of mol.: 103 / 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: 1.91 Å3/Da / Density % sol: 35.57 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 6.3
Details: 0.2M NH4Cl and 20% PEG 3350, pH 6.3, VAPOR DIFFUSION, HANGING DROP, temperature 295K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 HF / Wavelength: 1.5418 Å
DetectorType: RIGAKU RAXIS IV++ / Detector: IMAGE PLATE / Date: Aug 31, 2011
RadiationMonochromator: Mirror / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.19→40 Å / Num. all: 13511 / Num. obs: 13133 / % possible obs: 97.2 % / Observed criterion σ(F): -1 / Observed criterion σ(I): -1 / Redundancy: 3.7 % / Rmerge(I) obs: 0.107 / Rsym value: 0.107 / Net I/σ(I): 10.5
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique allRsym value% possible all
2.2-2.243.30.4442.56430.44497
2.24-2.283.30.4162.66460.41697.9
2.28-2.323.40.38336360.38397
2.32-2.373.40.363.26560.3696.9
2.37-2.423.40.3353.66170.33596.4
2.42-2.483.40.29946390.29995.9

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Processing

Software
NameVersionClassification
CrystalCleardata collection
MOLREPfrom ccp4phasing
REFMAC5.5.0109refinement
HKL-2000data reduction
HKL-2000data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 1SHH

1shh


Resolution: 2.19→29.13 Å / Cor.coef. Fo:Fc: 0.948 / Cor.coef. Fo:Fc free: 0.914 / SU B: 18.053 / SU ML: 0.204 / Cross valid method: THROUGHOUT / ESU R: 0.405 / ESU R Free: 0.257 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.2591 646 5 %RANDOM
Rwork0.19607 ---
obs0.19911 12369 95.95 %-
all-12891 --
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso mean: 41.076 Å2
Baniso -1Baniso -2Baniso -3
1-0.18 Å20 Å20 Å2
2---0.16 Å20 Å2
3----0.03 Å2
Refinement stepCycle: LAST / Resolution: 2.19→29.13 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2275 0 1 103 2379
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0120.0222333
X-RAY DIFFRACTIONr_angle_refined_deg1.4861.9623150
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.9495279
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.64423.091110
X-RAY DIFFRACTIONr_dihedral_angle_3_deg18.4815416
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.6161521
X-RAY DIFFRACTIONr_chiral_restr0.1070.2326
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.0211777
X-RAY DIFFRACTIONr_mcbond_it0.7591.51401
X-RAY DIFFRACTIONr_mcangle_it1.43222253
X-RAY DIFFRACTIONr_scbond_it1.9783932
X-RAY DIFFRACTIONr_scangle_it3.3114.5897
LS refinement shellResolution: 2.194→2.251 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.353 54 -
Rwork0.251 810 -
obs-810 88.16 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
13.2699-2.5797-1.993616.30974.95752.0181-0.608-0.70680.09421.10110.39940.98170.52720.38780.20860.36340.15520.07770.285-0.02640.0925-26.57324.236-6.377
21.05071.03971.41221.03811.40313.003-0.1652-0.02550.2589-0.1284-0.06080.2933-0.0617-0.08080.22590.17620.06820.00750.2775-0.00440.3-29.45626.71-22.241
30.6768-0.3694-0.16871.385-0.14320.1228-0.0796-0.1214-0.01010.13390.0655-0.1451-0.07910.00170.01410.23970.0252-0.04390.2574-0.00720.2257-6.14818.41-10.21
41.4748-1.3015-0.31277.84882.48620.8014-0.0982-0.29270.1460.17430.2237-0.52670.05770.0589-0.12550.28590.0546-0.01250.2640.0020.1416-7.18620.763-2.637
50.5064-0.3728-0.93231.32430.65961.9248-0.0795-0.0711-0.14340.1234-0.0642-0.0706-0.06790.14470.14370.25170.0094-0.00390.252-0.00920.2537-8.3888.251-12.694
60.5509-0.05030.55020.9611-0.45220.8776-0.0750.085-0.00540.10450.1228-0.01810.0119-0.0447-0.04780.19650.0594-0.00950.2588-0.01280.2224-19.71319.143-17.976
71.91812.26581.5825.67293.87764.11330.05920.0759-0.0934-0.14570.1029-0.2785-0.4404-0.1386-0.1620.21880.01130.0190.1980.01220.2092-13.35425.811-22.558
812.3814-5.16914.01393.6231-3.5163.61930.51940.062-0.2334-0.4731-0.07320.38840.47860.121-0.44620.20940.0187-0.06780.2311-0.04320.2454-11.0319.471-36.004
90.68220.1719-0.06830.7943-0.0620.4746-0.00460.0157-0.067-0.02320.0164-0.0172-0.0794-0.0462-0.01180.1950.0246-0.01130.2536-0.00170.2326-15.66217.995-25.045
100.0709-0.18250.7216.5008-0.20258.5202-0.002-0.025-0.04250.3481-0.02930.4480.4702-0.20920.03140.1681-0.00520.06620.26970.10870.3076-16.6192.034-8.992
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 10
2X-RAY DIFFRACTION2A11 - 15
3X-RAY DIFFRACTION3B16 - 72
4X-RAY DIFFRACTION4B73 - 91
5X-RAY DIFFRACTION5B92 - 115
6X-RAY DIFFRACTION6B116 - 146
7X-RAY DIFFRACTION7B150 - 164
8X-RAY DIFFRACTION8B165 - 175
9X-RAY DIFFRACTION9B176 - 232
10X-RAY DIFFRACTION10B233 - 247

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