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- PDB-1kzi: Crystal Structure of EcTS/dUMP/THF Complex -

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Basic information

Entry
Database: PDB / ID: 1kzi
TitleCrystal Structure of EcTS/dUMP/THF Complex
ComponentsThymidylate synthase
KeywordsTRANSFERASE / Enzyme substrate 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/dCMP hydroxymethylase / Thymidylate synthase, active site / Thymidylate synthase active site. / Thymidylate synthase / Thymidylate synthase/dCMP hydroxymethylase domain / Thymidylate synthase/dCMP hydroxymethylase superfamily / Thymidylate synthase / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
CARBONATE ION / 2,3-DIHYDROXY-1,4-DITHIOBUTANE / (2R,3S)-1,4-DIMERCAPTOBUTANE-2,3-DIOL / (6S)-5,6,7,8-TETRAHYDROFOLATE / 2'-DEOXYURIDINE 5'-MONOPHOSPHATE / Thymidylate synthase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.75 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 1.3Aug 16, 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 / 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
Remark 600 HETEROGEN THG 303 IS ASSOCIATED WITH CHAIN A. THG 305 IS ASSOCIATED WITH CHAIN B.

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)63,20712
Polymers61,1192
Non-polymers2,08810
Water6,503361
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8800 Å2
ΔGint-18 kcal/mol
Surface area19810 Å2
MethodPISA
Unit cell
Length a, b, c (Å)125.296, 125.296, 66.771
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number173
Space group name H-MP63
DetailsThe dimer comprising the asymmetric unit is the functional biological entity.

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Thymidylate synthase / / TS / TSase


Mass: 30559.666 Da / Num. of mol.: 2
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

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Non-polymers , 7 types, 371 molecules

#2: Chemical ChemComp-CO3 / CARBONATE ION / Carbonate


Mass: 60.009 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: CO3
#3: Chemical ChemComp-UMP / 2'-DEOXYURIDINE 5'-MONOPHOSPHATE / DUMP / Deoxyuridine monophosphate


Mass: 308.182 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C9H13N2O8P
#4: Chemical ChemComp-THG / (6S)-5,6,7,8-TETRAHYDROFOLATE


Mass: 445.429 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C19H23N7O6
#5: Chemical ChemComp-DTU / (2R,3S)-1,4-DIMERCAPTOBUTANE-2,3-DIOL / Dithioerythritol


Mass: 154.251 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O2S2
#6: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#7: Chemical ChemComp-DTT / 2,3-DIHYDROXY-1,4-DITHIOBUTANE / 1,4-DITHIOTHREITOL / Dithiothreitol


Mass: 154.251 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O2S2
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 361 / 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: 2.47 Å3/Da / Density % sol: 50.29 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 8.6
Details: 100 mM TAPS, 2.6 M ammonium sulfate, 10 mM DTT, pH 8.6, VAPOR DIFFUSION, HANGING DROP, temperature 298K
Crystal grow
*PLUS
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDDetails
16.1 mg/mlEcTS1drop
22 mMTHF1drop
31 mMdUMP1drop
45 mMdithiothreitol1drop
50.9 mMEDTA1drop
6100 mMTAPS buffer1reservoirpH8.6
72.6 Mammonium sulfate1reservoir
810 mMdithiothreitol1reservoir

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU / Wavelength: 1.5418 Å
DetectorType: RIGAKU RAXIS IV / Detector: IMAGE PLATE / Date: Aug 16, 2000 / Details: Osmic mirrors
RadiationMonochromator: Osmic mirrors / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.75→29.46 Å / Num. all: 60506 / Num. obs: 59657 / % possible obs: 98.6 % / Observed criterion σ(I): -3 / Biso Wilson estimate: 25.9 Å2
Reflection shellResolution: 1.75→1.81 Å / % possible all: 88.1
Reflection
*PLUS
Num. measured all: 492649 / Rmerge(I) obs: 0.043
Reflection shell
*PLUS
% possible obs: 88.1 % / Rmerge(I) obs: 0.454 / Mean I/σ(I) obs: 2

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Processing

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

2kce


Resolution: 1.75→29.46 Å / Rfactor Rfree error: 0.004 / Data cutoff high absF: 1912305.65 / 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
Rfree0.221 3007 5.1 %RANDOM
Rwork0.197 ---
obs-59490 98.6 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 43.3651 Å2 / ksol: 0.375416 e/Å3
Displacement parametersBiso mean: 27.3 Å2
Baniso -1Baniso -2Baniso -3
1--1.06 Å20.33 Å20 Å2
2---1.06 Å20 Å2
3---2.13 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.24 Å0.21 Å
Luzzati d res low-5 Å
Luzzati sigma a0.19 Å0.17 Å
Refinement stepCycle: LAST / Resolution: 1.75→29.46 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4306 0 138 361 4805
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.007
X-RAY DIFFRACTIONc_angle_deg1.5
X-RAY DIFFRACTIONc_dihedral_angle_d24.1
X-RAY DIFFRACTIONc_improper_angle_d0.9
LS refinement shellResolution: 1.75→1.86 Å / Rfactor Rfree error: 0.017 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.375 467 5.1 %
Rwork0.345 8758 -
obs--92.4 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2FORM_DUMP_THF.PARAMFORM_DUMP_THF.TOPO
X-RAY DIFFRACTION3DTT_ODT_GCL.PARAMDTT_ODT_GCL.TOPO
X-RAY DIFFRACTION4ION.PARAMION.TOPO
X-RAY DIFFRACTION5WATER_REP.PARAMWATER.TOP
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.007
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg24.1
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg0.9
LS refinement shell
*PLUS
Lowest resolution: 1.81 Å / Rfactor Rfree: 0.42 / Rfactor Rwork: 0.391

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