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- PDB-1kzj: Crystal Structure of EcTS W80G/dUMP/CB3717 Complex -

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Basic information

Entry
Database: PDB / ID: 1kzj
TitleCrystal Structure of EcTS W80G/dUMP/CB3717 Complex
ComponentsThymidylate synthase
KeywordsTRANSFERASE / Enzyme substrate cofactor analog complex
Function / homology
Function and homology information


thymidylate synthase / thymidylate synthase activity / dTMP biosynthetic process / dTTP biosynthetic process / response to radiation / regulation of translation / methylation / magnesium ion binding / protein homodimerization activity / RNA binding / cytosol
Similarity search - Function
Thymidylate Synthase; Chain A / Thymidylate synthase/dCMP hydroxymethylase domain / Thymidylate synthase, active site / Thymidylate synthase active site. / Thymidylate synthase / Thymidylate synthase/dCMP hydroxymethylase / Thymidylate synthase/dCMP hydroxymethylase domain / Thymidylate synthase/dCMP hydroxymethylase superfamily / Thymidylate synthase / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
10-PROPARGYL-5,8-DIDEAZAFOLIC ACID / 2'-DEOXYURIDINE 5'-MONOPHOSPHATE / Thymidylate synthase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.6 Å
AuthorsFritz, T.A. / Liu, L. / Finer-Moore, J.S. / Stroud, R.M.
CitationJournal: Biochemistry / Year: 2002
Title: Tryptophan 80 and leucine 143 are critical for the hydride transfer step of thymidylate synthase by controlling active site access.
Authors: Fritz, T.A. / Liu, L. / Finer-Moore, J.S. / Stroud, R.M.
History
DepositionFeb 6, 2002Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 3, 2002Provider: repository / Type: Initial release
Revision 1.1Apr 28, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Non-polymer description / Version format compliance
Revision 1.3Oct 27, 2021Group: Database references / Derived calculations
Category: database_2 / struct_conn ...database_2 / 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 / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _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_asym_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_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.4Aug 16, 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: Thymidylate synthase
B: Thymidylate synthase
C: Thymidylate synthase
D: Thymidylate synthase
E: Thymidylate synthase
F: Thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)187,29718
Polymers182,5836
Non-polymers4,71412
Water5,260292
1
A: Thymidylate synthase
B: Thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)62,4326
Polymers60,8612
Non-polymers1,5714
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7880 Å2
ΔGint-24 kcal/mol
Surface area19510 Å2
MethodPISA
2
C: Thymidylate synthase
D: Thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)62,4326
Polymers60,8612
Non-polymers1,5714
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7710 Å2
ΔGint-23 kcal/mol
Surface area19580 Å2
MethodPISA
3
E: Thymidylate synthase
F: Thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)62,4326
Polymers60,8612
Non-polymers1,5714
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7680 Å2
ΔGint-19 kcal/mol
Surface area19920 Å2
MethodPISA
Unit cell
Length a, b, c (Å)95.545, 84.574, 111.522
Angle α, β, γ (deg.)90.00, 111.37, 90.00
Int Tables number4
Space group name H-MP1211
DetailsThe biological unit consists of a dimer comprised of chains A and B or C and D or E and F of the asymmetric unit.

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Components

#1: Protein
Thymidylate synthase / TS / TSase


Mass: 30430.506 Da / Num. of mol.: 6 / Mutation: W80G
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) / References: UniProt: P0A884, thymidylate synthase
#2: Chemical
ChemComp-UMP / 2'-DEOXYURIDINE 5'-MONOPHOSPHATE / DUMP


Mass: 308.182 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C9H13N2O8P
#3: Chemical
ChemComp-CB3 / 10-PROPARGYL-5,8-DIDEAZAFOLIC ACID


Mass: 477.469 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C24H23N5O6
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 292 / 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: 2.3 Å3/Da / Density % sol: 46.45 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: 0.1 M Tris, 22-26% PEG 4000, 0.2 M sodium acetate, 10 mM DTT, pH 8.5, VAPOR DIFFUSION, HANGING DROP, temperature 298K
Crystal grow
*PLUS
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDDetails
13-5 mg/mlprotein1drop
22 mMdUMP1drop
31 mMCB37171drop
45 mMdithiothreitol1drop
50.1 MTris1reservoirpH8.5
622-26 %(w/v)PEG40001reservoir
70.2 Msodium acetate1reservoir
810 mMdithiothreitol1reservoir

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL7-1 / Wavelength: 1.08 Å
DetectorType: MARRESEARCH / Detector: IMAGE PLATE / Date: Nov 20, 1999
RadiationMonochromator: Cylindrically bent, triangular, Si 111 asymmetric cut, horizontal focus
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.08 Å / Relative weight: 1
ReflectionResolution: 2.6→25.35 Å / Num. obs: 51013 / % possible obs: 98.1 % / Observed criterion σ(I): -3 / Biso Wilson estimate: 26.3 Å2
Reflection shellResolution: 2.6→2.69 Å / % possible all: 92.4
Reflection
*PLUS
Highest resolution: 2.6 Å / Num. measured all: 444846 / Rmerge(I) obs: 0.124
Reflection shell
*PLUS
% possible obs: 92.4 % / Rmerge(I) obs: 0.41 / Mean I/σ(I) obs: 2.4

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Processing

Software
NameVersionClassification
EPMRphasing
CNS1.1refinement
DENZOdata reduction
SCALEPACKdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 2KCE with ligands and water removed.

2kce


Resolution: 2.6→25.35 Å / Rfactor Rfree error: 0.004 / Data cutoff high absF: 1552392.29 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.258 3545 7.1 %RANDOM
Rwork0.215 ---
obs-50201 98.2 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 27.3867 Å2 / ksol: 0.350189 e/Å3
Displacement parametersBiso mean: 20.8 Å2
Baniso -1Baniso -2Baniso -3
1--4.38 Å20 Å2-2.48 Å2
2---1.19 Å20 Å2
3---5.57 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.39 Å0.31 Å
Luzzati d res low-5 Å
Luzzati sigma a0.48 Å0.35 Å
Refinement stepCycle: LAST / Resolution: 2.6→25.35 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms12837 0 330 292 13459
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.008
X-RAY DIFFRACTIONc_angle_deg1.5
X-RAY DIFFRACTIONc_dihedral_angle_d24.6
X-RAY DIFFRACTIONc_improper_angle_d1.18
LS refinement shellResolution: 2.6→2.76 Å / Rfactor Rfree error: 0.015 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.347 567 7.2 %
Rwork0.283 7298 -
obs--93.2 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2DUMP_CB3717.PARAMDUMP_CB3717.TOPO
X-RAY DIFFRACTION3FORM.PARAMFORM.TOPO
X-RAY DIFFRACTION4WATER_REP.PARAMWATER.TOP
Refinement
*PLUS
Highest resolution: 2.6 Å / Rfactor Rfree: 0.258 / Rfactor Rwork: 0.215
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.008
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg24.6
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg1.18
LS refinement shell
*PLUS
Lowest resolution: 2.69 Å / Rfactor Rfree: 0.34 / Rfactor Rwork: 0.28

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