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- PDB-3ee0: Crystal Structure of the W215A/E217A Mutant of Human Thrombin (sp... -

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Basic information

Entry
Database: PDB / ID: 3ee0
TitleCrystal Structure of the W215A/E217A Mutant of Human Thrombin (space group P2(1)2(1)2(1))
Components
  • Thrombin heavy chain
  • Thrombin light chain
KeywordsHYDROLASE / Serine protease / Acute phase / Blood coagulation / Calcium / Cleavage on pair of basic residues / Disease mutation / Gamma-carboxyglutamic acid / Glycoprotein / Kringle / Pharmaceutical / Polymorphism / Protease / Secreted / Zymogen
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: 2.75 Å
AuthorsGandhi, P.S. / Page, M.J. / Chen, Z. / Bush-Pelc, L. / Di Cera, E.
Citation
Journal: To be Published
Title: Molecular Basis for the Kinetic Differences of the W215A/E217A Mutant of Human and Murine Thrombin
Authors: Gandhi, P.S. / Page, M.J. / Chen, Z. / Bush-Pelc, L. / Di Cera, E.
#1: Journal: J.Biol.Chem. / Year: 2004
Title: The Anticoagulant Thrombin Mutant W215A/E217A Has a Collapsed Primary Specificity Pocket
Authors: Pineda, A.O. / Chen, Z. / Caccia, S. / Cantwell, A.M. / Savvides, S.N. / Waksman, G. / Mathews, F.S. / Di Cera, E.
History
DepositionSep 3, 2008Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 7, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Oct 20, 2021Group: Database references / Category: database_2 / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details
Revision 1.3Aug 30, 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: Thrombin light chain
B: Thrombin heavy chain


Theoretical massNumber of molelcules
Total (without water)33,7042
Polymers33,7042
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2200 Å2
ΔGint-11 kcal/mol
Surface area12850 Å2
MethodPISA
Unit cell
Length a, b, c (Å)40.142, 60.007, 120.011
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 genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F2 / Plasmid: HPC4-pNUT / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P00734, thrombin
#2: Protein Thrombin heavy chain /


Mass: 29607.055 Da / Num. of mol.: 1 / Mutation: W215A, E217A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F2 / Plasmid: HPC4-pNUT / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P00734, thrombin

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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: 2.14 Å3/Da / Density % sol: 42.64 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 0.1M MES, 30% PEG 400, pH 6.5, VAPOR DIFFUSION, HANGING DROP, temperature 295K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 14-BM-C / Wavelength: 0.9 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Feb 29, 2008
RadiationMonochromator: bent Ge(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9 Å / Relative weight: 1
ReflectionResolution: 2.7→40 Å / Num. all: 8496 / Num. obs: 8182 / % possible obs: 96.3 % / Observed criterion σ(F): -1 / Observed criterion σ(I): -1 / Redundancy: 5.4 % / Biso Wilson estimate: 24.3 Å2 / Rmerge(I) obs: 0.089 / Net I/σ(I): 14.7
Reflection shellResolution: 2.7→2.8 Å / Redundancy: 2.8 % / Rmerge(I) obs: 0.332 / Mean I/σ(I) obs: 2.5 / Num. unique all: 636 / % possible all: 78.3

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Processing

Software
NameVersionClassification
ADSCQuantumdata collection
MOLREPfrom ccp4phasing
CNS1.2refinement
HKL-2000data reduction
SCALEPACKdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 1TQ0

1tq0


Resolution: 2.75→26.84 Å / Rfactor Rfree error: 0.016 / Data cutoff high absF: 83494.39 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / σ(I): 0 / Stereochemistry target values: Engh & Huber / Details: BULK SOLVENT MODEL USED
RfactorNum. reflection% reflectionSelection details
Rfree0.323 418 5.4 %RANDOM
Rwork0.239 ---
obs0.239 7683 96.2 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 45.9724 Å2 / ksol: 0.4 e/Å3
Displacement parametersBiso mean: 38.4 Å2
Baniso -1Baniso -2Baniso -3
1--3.6 Å20 Å20 Å2
2---2.33 Å20 Å2
3---5.93 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.54 Å0.36 Å
Luzzati d res low-5 Å
Luzzati sigma a0.56 Å0.41 Å
Refinement stepCycle: LAST / Resolution: 2.75→26.84 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2217 0 0 0 2217
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.008
X-RAY DIFFRACTIONc_angle_deg1.3
X-RAY DIFFRACTIONc_dihedral_angle_d23.9
X-RAY DIFFRACTIONc_improper_angle_d0.81
LS refinement shellResolution: 2.75→2.92 Å / Rfactor Rfree error: 0.044 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.374 72 6.6 %
Rwork0.315 1018 -
obs--84.3 %
Xplor fileSerial no: 1 / Param file: protein_rep.param / Topol file: protein.top

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