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- PDB-1ffl: CRYSTAL STRUCTURE OF THE APO-THYMIDYLATE SYNTHASE R166Q MUTANT -

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Basic information

Entry
Database: PDB / ID: 1ffl
TitleCRYSTAL STRUCTURE OF THE APO-THYMIDYLATE SYNTHASE R166Q MUTANT
ComponentsTHYMIDYLATE SYNTHASE
KeywordsTRANSFERASE / METHYLTRANSFERASE
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
Thymidylate synthase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / Fourier Difference Maps / Resolution: 2.94 Å
AuthorsSotelo-Mundo, R.R. / Changchien, L. / Maley, F. / Montfort, W.R.
Citation
Journal: J.Biochem.Mol.Toxicol. / Year: 2006
Title: Crystal structures of thymidylate synthase mutant R166Q: Structural basis for the nearly complete loss of catalytic activity.
Authors: Sotelo-Mundo, R.R. / Changchien, L. / Maley, F. / Montfort, W.R.
#1: Journal: Thesis / Year: 1999
Title: Crystallographic Studies of Thymidylate Synthase : Structure of a Mammalian Enzyme and Analyses of Invariant Non-Catalytic Residues in a Bacterial Enzyme.
Authors: Sotelo-Mundo, R.R.
History
DepositionJul 25, 2000Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 28, 2000Provider: repository / Type: Initial release
Revision 1.1Apr 27, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Derived calculations / Version format compliance
Revision 1.3Oct 4, 2017Group: Refinement description / Category: software / Item: _software.name
Revision 1.4Apr 18, 2018Group: Data collection / Category: diffrn_detector / Item: _diffrn_detector.detector
Revision 1.5Nov 3, 2021Group: Database references / Derived calculations / Category: database_2 / struct_conn / struct_ref_seq_dif
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
Revision 1.6Apr 3, 2024Group: 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: THYMIDYLATE SYNTHASE


Theoretical massNumber of molelcules
Total (without water)30,5311
Polymers30,5311
Non-polymers00
Water18010
1
A: THYMIDYLATE SYNTHASE

A: THYMIDYLATE SYNTHASE


Theoretical massNumber of molelcules
Total (without water)61,0612
Polymers61,0612
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation16_555x,-y,-z+1/21
Buried area4270 Å2
ΔGint-20 kcal/mol
Surface area21200 Å2
MethodPISA, PQS
Unit cell
Length a, b, c (Å)132.500, 132.500, 132.500
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number199
Space group name H-MI213
Components on special symmetry positions
IDModelComponents
11A-300-

HOH

DetailsThe biological assembly is a dimer constructed from chain A a symmetry partner.

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Components

#1: Protein THYMIDYLATE SYNTHASE /


Mass: 30530.600 Da / Num. of mol.: 1 / Mutation: R166Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Plasmid: BLUESCRIPT PLASMID SK+ / Production host: Escherichia coli (E. coli)
Strain (production host): XAC 25: LACKS HOST THYMIDYLATE SYNTHASE GENE
References: UniProt: P0A884, thymidylate synthase
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 10 / 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: 3.17 Å3/Da / Density % sol: 61.24 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 8.1
Details: Ammonium sulfate, potassium phosphate, EDTA, DTT, pH 8.1, VAPOR DIFFUSION, HANGING DROP, temperature 298K
Crystal grow
*PLUS
pH: 8 / Method: vapor diffusion / Details: Montfort, W.R., (1990) Biochemistry, 29, 6964.
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-ID
15-9 mg/mLprotein1drop
220 mMpotasssium1drop
32.3 Mammonium1drop
4100 mMdUMP1drop
54.78 mMCB37171drop
620 mMpotassium1reservoir
70.1 mMdisodium ethylenediaminetetraacetic acid1reservoir
820 mMbeta-mercaptoethanol1reservoir

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Data collection

DiffractionMean temperature: 298 K
Diffraction sourceSource: ROTATING ANODE / Type: ENRAF-NONIUS FR571 / Wavelength: 1.5418
DetectorType: ENRAF-NONIUS FAST / Detector: DIFFRACTOMETER / Date: May 4, 1997
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.94→35.335 Å / Num. all: 8138 / Num. obs: 8138 / % possible obs: 96.1 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 7 % / Biso Wilson estimate: 0 Å2 / Rmerge(I) obs: 0.09 / Net I/σ(I): 7.4
Reflection shellResolution: 2.94→3.02 Å / Redundancy: 6.1 % / Rmerge(I) obs: 0.38 / Num. unique all: 610 / % possible all: 96
Reflection
*PLUS
Lowest resolution: 34 Å / % possible obs: 96 %
Reflection shell
*PLUS
% possible obs: 96 % / Mean I/σ(I) obs: 1.9

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Processing

Software
NameVersionClassification
CNSrefinement
MADNESSdata reduction
CCP4(AGROVATAdata scaling
ROTAVATAdata scaling
CNSphasing
RefinementMethod to determine structure: Fourier Difference Maps
Starting model: Wildtype Binary Complex of E.coli thymidylate synthase

Resolution: 2.94→33.13 Å / Rfactor Rfree error: 0.007 / Data cutoff high absF: 3374507.52 / Data cutoff low absF: 0 / Isotropic thermal model: GROUP / Cross valid method: THROUGHOUT / σ(F): 0.0001 / σ(I): 3 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.22 868 10.7 %RANDOM
Rwork0.171 ---
all0.171 8390 --
obs0.171 8138 97 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 28.17 Å2 / ksol: 0.308 e/Å3
Displacement parametersBiso mean: 39.5 Å2
Baniso -1Baniso -2Baniso -3
1-0 Å20 Å20 Å2
2--0 Å20 Å2
3---0 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.35 Å0.27 Å
Luzzati d res low-5 Å
Luzzati sigma a0.43 Å0.38 Å
Refinement stepCycle: LAST / Resolution: 2.94→33.13 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2151 0 0 10 2161
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.007
X-RAY DIFFRACTIONc_angle_deg1.3
X-RAY DIFFRACTIONc_dihedral_angle_d24.6
X-RAY DIFFRACTIONc_improper_angle_d0.81
LS refinement shellResolution: 2.94→3.12 Å / Rfactor Rfree error: 0.027 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.311 137 10.7 %
Rwork0.258 1147 -
obs--93.3 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2CBX.PARWATER.TOP
X-RAY DIFFRACTION3WATER_REP.PARAM
Software
*PLUS
Name: CNS / Version: 1 / Classification: refinement
Refinement
*PLUS
σ(F): 0 / % reflection Rfree: 10.7 % / Rfactor obs: 0.17 / Rfactor Rfree: 0.21
Solvent computation
*PLUS
Displacement parameters
*PLUS
Biso mean: 39.5 Å2
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.008
X-RAY DIFFRACTIONc_angle_deg1.4
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg24.6
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg0.81
LS refinement shell
*PLUS
Rfactor Rfree: 0.311 / % reflection Rfree: 10.7 % / Rfactor Rwork: 0.258

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