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- PDB-3edx: Crystal structure of the W215A/E217A mutant of murine thrombin -

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Basic information

Entry
Database: PDB / ID: 3edx
TitleCrystal structure of the W215A/E217A mutant of murine thrombin
Components
  • Thrombin heavy chain
  • Thrombin light chain
KeywordsHYDROLASE / Serine protease / Acute phase / Blood coagulation / Cleavage on pair of basic residues / Gamma-carboxyglutamic acid / Glycoprotein / Kringle / Protease / Zymogen
Function / homology
Function and homology information


Common Pathway of Fibrin Clot Formation / Platelet Aggregation (Plug Formation) / Gamma-carboxylation of protein precursors / Transport of gamma-carboxylated protein precursors from the endoplasmic reticulum to the Golgi apparatus / Intrinsic Pathway of Fibrin Clot Formation / Removal of aminoterminal propeptides from gamma-carboxylated proteins / thrombin-activated receptor signaling pathway / Peptide ligand-binding receptors / Thrombin signalling through proteinase activated receptors (PARs) / Regulation of Complement cascade ...Common Pathway of Fibrin Clot Formation / Platelet Aggregation (Plug Formation) / Gamma-carboxylation of protein precursors / Transport of gamma-carboxylated protein precursors from the endoplasmic reticulum to the Golgi apparatus / Intrinsic Pathway of Fibrin Clot Formation / Removal of aminoterminal propeptides from gamma-carboxylated proteins / thrombin-activated receptor signaling pathway / Peptide ligand-binding receptors / Thrombin signalling through proteinase activated receptors (PARs) / Regulation of Complement cascade / G alpha (q) signalling events / Cell surface interactions at the vascular wall / positive regulation of phospholipase C-activating G protein-coupled receptor signaling pathway / cytolysis by host of symbiont cells / thrombospondin receptor activity / thrombin / neutrophil-mediated killing of gram-negative bacterium / ligand-gated ion channel signaling pathway / 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 / fibrinolysis / regulation of cytosolic calcium ion concentration / positive regulation of release of sequestered calcium ion into cytosol / acute-phase response / lipopolysaccharide binding / negative regulation of proteolysis / positive regulation of insulin secretion / platelet activation / response to wounding / positive regulation of protein localization to nucleus / positive regulation of reactive oxygen species metabolic process / antimicrobial humoral immune response mediated by antimicrobial peptide / heparin binding / peptidase activity / regulation of cell shape / positive regulation of cell growth / regulation of gene expression / collagen-containing extracellular matrix / endopeptidase activity / 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 / external side of plasma membrane / signaling receptor binding / serine-type endopeptidase activity / calcium ion binding / positive regulation of cell population proliferation / proteolysis / extracellular space
Similarity search - Function
Epsilon-Thrombin; Chain L / Thrombin light chain domain / Prothrombin/thrombin / Thrombin light chain / Thrombin light chain domain superfamily / Thrombin light chain / Kringle domain / Kringle / Kringle, conserved site / Kringle superfamily ...Epsilon-Thrombin; Chain L / Thrombin light chain domain / 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 / Few Secondary Structures / Irregular / 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 speciesMus musculus (house mouse)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.4 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 W215/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: Non-polymer description / Version format compliance
Revision 1.2Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_conn / struct_site / struct_site_gen
Item: _chem_comp.name / _chem_comp.type ..._chem_comp.name / _chem_comp.type / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.pdbx_role
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 1.3Oct 20, 2021Group: Database references / Structure summary / Category: chem_comp / database_2 / struct_ref_seq_dif
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI ..._chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details
Revision 1.4Aug 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

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Assembly

Deposited unit
A: Thrombin light chain
B: Thrombin heavy chain
C: Thrombin light chain
D: Thrombin heavy chain
E: Thrombin light chain
F: Thrombin heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)106,04016
Polymers104,7046
Non-polymers1,33610
Water4,089227
1
A: Thrombin light chain
B: Thrombin heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)35,3155
Polymers34,9012
Non-polymers4133
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3790 Å2
ΔGint-45 kcal/mol
Surface area14070 Å2
MethodPISA
2
C: Thrombin light chain
D: Thrombin heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)35,4116
Polymers34,9012
Non-polymers5094
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3900 Å2
ΔGint-51 kcal/mol
Surface area14070 Å2
MethodPISA
3
E: Thrombin light chain
F: Thrombin heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)35,3155
Polymers34,9012
Non-polymers4133
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3840 Å2
ΔGint-43 kcal/mol
Surface area13920 Å2
MethodPISA
Unit cell
Length a, b, c (Å)97.716, 105.760, 176.811
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: 5105.731 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: F2, Cf2 / Plasmid: HPC4-pNUT / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P19221, thrombin
#2: Protein Thrombin heavy chain /


Mass: 29795.461 Da / Num. of mol.: 3 / Mutation: W215A, E217A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: F2, Cf2 / Plasmid: HPC4-pNUT / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: P19221, thrombin
#3: Chemical
ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: SO4
#4: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 227 / 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: 4.36 Å3/Da / Density % sol: 71.81 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 6.6
Details: 0.2M NA2SO4, 20 % PEG 3350, pH 6.6, 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: Apr 14, 2006
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9 Å / Relative weight: 1
ReflectionResolution: 2.4→40 Å / Num. all: 71558 / Num. obs: 67622 / % possible obs: 94.5 % / Observed criterion σ(F): -1 / Observed criterion σ(I): -1 / Redundancy: 5.6 % / Biso Wilson estimate: 33.3 Å2 / Rmerge(I) obs: 0.059 / Net I/σ(I): 21.2
Reflection shellResolution: 2.4→2.49 Å / Redundancy: 4.1 % / Rmerge(I) obs: 0.461 / Mean I/σ(I) obs: 2.5 / Num. unique all: 5642 / % possible all: 79.9

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Processing

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

1tq0


Resolution: 2.4→32.78 Å / Rfactor Rfree error: 0.004 / Data cutoff high absF: 219529.95 / Data cutoff low absF: 0 / Isotropic thermal model: Isotropic / Cross valid method: THROUGHOUT / σ(F): 0 / σ(I): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.243 3371 5.1 %RANDOM
Rwork0.214 ---
obs0.214 66166 92.2 %-
all-72302 --
Displacement parametersBiso mean: 55.6 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.38 Å0.32 Å
Luzzati d res low-5 Å
Luzzati sigma a0.5 Å0.45 Å
Refinement stepCycle: LAST / Resolution: 2.4→32.78 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7308 0 77 227 7612
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.007
X-RAY DIFFRACTIONc_angle_deg1.4
X-RAY DIFFRACTIONc_dihedral_angle_d23.9
X-RAY DIFFRACTIONc_improper_angle_d0.87
LS refinement shellResolution: 2.4→2.55 Å / Rfactor Rfree error: 0.016 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.345 445 5.2 %
Rwork0.33 8125 -
obs-8478 72.3 %

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