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- PDB-1nce: Crystal structure of a ternary complex of E. coli thymidylate syn... -

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Basic information

Entry
Database: PDB / ID: 1nce
TitleCrystal structure of a ternary complex of E. coli thymidylate synthase D169C with dUMP and the antifolate CB3717
ComponentsThymidylate synthase
KeywordsBIOSYNTHETIC PROTEIN / beta-sheet interface / protein-dUMP-cofactor analog complex / asymmetric dimer
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
10-PROPARGYL-5,8-DIDEAZAFOLIC ACID / 2'-DEOXYURIDINE 5'-MONOPHOSPHATE / Thymidylate synthase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Escherichia coli O157:H7 (bacteria)
MethodX-RAY DIFFRACTION / FOURIER SYNTHESIS / Resolution: 2.4 Å
AuthorsBirdsall, D.L. / Finer-Moore, J. / Stroud, R.M.
Citation
Journal: Protein Eng. / Year: 2003
Title: The only active mutant of thymidylate synthase D169, a residue far from the site of methyl transfer, demonstrates the exquisite nature of enzyme specificity.
Authors: Birdsall, D.L. / Finer-Moore, J. / Stroud, R.M.
#1: Journal: Biochemistry / Year: 1998
Title: D221 in thymidylate synthase controls conformation change, and thereby opening of the imidazolidine
Authors: Sage, C.R. / Michelitsch, M.D. / Stout, T.J. / Biermann, D. / Nissen, R. / Finer-Moore, J. / Stroud, R.M.
#2: Journal: Biochemistry / Year: 1990
Title: Structure, multiple site binding, and segmental accommodation in thymidylate synthase on binding dUMP and an anti-folate
Authors: Montfort, W.R. / Perry, K.M. / Fauman, E.B. / Finer-Moore, J.S. / Maley, G.F. / Hardy, L. / Maley, F. / Stroud, R.M.
History
DepositionDec 5, 2002Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 25, 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 1.3Jan 24, 2018Group: Database references / Category: citation_author / Item: _citation_author.name
Revision 1.4Oct 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_leaving_atom_flag / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.5Aug 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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)62,6676
Polymers61,0952
Non-polymers1,5714
Water3,207178
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area7950 Å2
ΔGint-19 kcal/mol
Surface area19290 Å2
MethodPISA
Unit cell
Length a, b, c (Å)127.100, 127.100, 68.100
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number173
Space group name H-MP63
DetailsThe biological assembly is a homodimer consisting of chains A and B

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Components

#1: Protein Thymidylate synthase / / TS / TSase


Mass: 30547.721 Da / Num. of mol.: 2 / Mutation: D169C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli, Escherichia coli O157:H7
Genus: Escherichia, Escherichia / Species: , Escherichia coli / Strain: , O157:H7 / Gene: THYA OR B2827 OR Z4144 OR ECS3684 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A884, thymidylate synthase
#2: Chemical ChemComp-UMP / 2'-DEOXYURIDINE 5'-MONOPHOSPHATE / DUMP / Deoxyuridine monophosphate


Mass: 308.182 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C9H13N2O8P
#3: Chemical ChemComp-CB3 / 10-PROPARGYL-5,8-DIDEAZAFOLIC ACID


Mass: 477.469 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C24H23N5O6
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 178 / 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: 2.6 Å3/Da / Density % sol: 52.65 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 9.1
Details: potassium phosphate, EDTA, ammonium sulfate, DTT, pH 9.1, VAPOR DIFFUSION, HANGING DROP, temperature 298.0K
Crystal grow
*PLUS
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-ID
113 mg/mlprotein1drop
210 mMpotassium phosphate1drop
350000 mMEDTA1drop
41.2 Mammonium sulfate1drop
510 mMdithiothreitol1drop
66 mMdUMP1drop
740000 mMCB37171drop
820 mMpotassium phosphate1reservoir
9100000 mMEDTA1reservoir
1010 mMdithiothreitol1reservoir
112.4 Mammonium sulfate1reservoir

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Data collection

DiffractionMean temperature: 298 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RU200 / Wavelength: 1.5418
DetectorType: RIGAKU RAXIS II / Detector: IMAGE PLATE / Date: Dec 21, 1998 / Details: monochromater
RadiationMonochromator: graphite / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.4→40 Å / Num. all: 24679 / Num. obs: 24679 / % possible obs: 100 % / Observed criterion σ(F): -3 / Observed criterion σ(I): -3 / Redundancy: 8.7 % / Rmerge(I) obs: 0.174 / Net I/σ(I): 8.1
Reflection shellResolution: 2.4→2.55 Å / Mean I/σ(I) obs: 1.9 / % possible all: 100
Reflection
*PLUS
% possible obs: 100 %

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Processing

Software
NameClassification
DENZOdata reduction
SCALEPACKdata scaling
CNSrefinement
CNSphasing
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: PDB entry 1KCE with waters and ligands removed

1kce


Resolution: 2.4→36.69 Å / Rfactor Rfree error: 0.006 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / σ(I): -3 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.246 1694 6.9 %RANDOM
Rwork0.187 ---
all0.1913 24679 --
obs0.187 24679 100 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 56.9659 Å2 / ksol: 0.334593 e/Å3
Displacement parametersBiso mean: 34.2 Å2
Baniso -1Baniso -2Baniso -3
1--1.95 Å20.26 Å20 Å2
2---1.95 Å20 Å2
3---3.9 Å2
Refine analyzeLuzzati coordinate error free: 0.37 Å / Luzzati sigma a free: 0.5 Å
Refinement stepCycle: LAST / Resolution: 2.4→36.69 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4300 0 110 178 4588
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.008
X-RAY DIFFRACTIONc_angle_deg1.6
X-RAY DIFFRACTIONc_dihedral_angle_d24.3
X-RAY DIFFRACTIONc_improper_angle_d0.94
X-RAY DIFFRACTIONc_mcbond_it3.151.5
X-RAY DIFFRACTIONc_mcangle_it4.712
X-RAY DIFFRACTIONc_scbond_it3.382
X-RAY DIFFRACTIONc_scangle_it5.092.5
LS refinement shellResolution: 2.4→2.55 Å / Rfactor Rfree error: 0.02 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.337 284 7 %
Rwork0.299 3779 -
obs--100 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2WATER_REP.PARAMWATER.TOP
X-RAY DIFFRACTION3PARAMED.LIGTOPO.DUMP
X-RAY DIFFRACTION4FORM.PARAMTOPO.CB3
X-RAY DIFFRACTION5FORM.TOPO
Refinement
*PLUS
Highest resolution: 2.4 Å
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg24.3
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg0.94
LS refinement shell
*PLUS
Highest resolution: 2.4 Å

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