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- PDB-1evg: CRYSTAL STRUCTURE ANALYSIS OF CYS167 MUTANT OF ESCHERICHIA COLI W... -

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Basic information

Entry
Database: PDB / ID: 1evg
TitleCRYSTAL STRUCTURE ANALYSIS OF CYS167 MUTANT OF ESCHERICHIA COLI WITH UNMODIFIED CATALYTIC CYSTEINE
ComponentsTHYMIDYLATE SYNTHASE
KeywordsTRANSFERASE / Thr167 E. coli Thymidylate Synthase with Unmodified Catalytic Cysteine
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 / Resolution: 2 Å
AuthorsPhan, J. / Mahdavian, E. / Nivens, M.C. / Minor, W. / Berger, S. / Spencer, H.T. / Dunlap, R.B. / Lebioda, L.
CitationJournal: Biochemistry / Year: 2000
Title: Catalytic cysteine of thymidylate synthase is activated upon substrate binding.
Authors: Phan, J. / Mahdavian, E. / Nivens, M.C. / Minor, W. / Berger, S. / Spencer, H.T. / Dunlap, R.B. / Lebioda, L.
History
DepositionApr 19, 2000Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 3, 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
Revision 1.4Jan 31, 2018Group: Experimental preparation / Category: exptl_crystal_grow
Item: _exptl_crystal_grow.pdbx_details / _exptl_crystal_grow.temp
Revision 1.5Nov 3, 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 1.6Apr 13, 2022Group: Database references / Structure summary / Category: audit_author / citation_author
Item: _audit_author.identifier_ORCID / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: THYMIDYLATE SYNTHASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)31,0144
Polymers30,7261
Non-polymers2883
Water1,54986
1
A: THYMIDYLATE SYNTHASE
hetero molecules

A: THYMIDYLATE SYNTHASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)62,0288
Polymers61,4522
Non-polymers5766
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation15_556-x+1/2,y,-z+11
Buried area5400 Å2
ΔGint-96 kcal/mol
Surface area21110 Å2
MethodPISA, PQS
Unit cell
Length a, b, c (Å)131.973, 131.973, 131.973
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-374-

HOH

21A-379-

HOH

31A-388-

HOH

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Components

#1: Protein THYMIDYLATE SYNTHASE /


Mass: 30725.924 Da / Num. of mol.: 1 / Mutation: S167T
Source method: isolated from a genetically manipulated source
Details: UNMODIFIED CATALYTIC CYSTEINE / Source: (gene. exp.) Escherichia coli (E. coli) / Plasmid: PBLUESCRIPT SK / Production host: Escherichia coli (E. coli) / References: UniProt: P0A884, thymidylate synthase
#2: Chemical ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: SO4
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 86 / 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: 3.12 Å3/Da / Density % sol: 60.52 %
Crystal growTemperature: 291 K / Method: vapor diffusion, hanging drop / pH: 7.8
Details: 48% saturated ammonium sulfate, 100 mM Tris HCl, 20 mM 2-mercaptoethanol , pH 7.8, VAPOR DIFFUSION, HANGING DROP, temperature 18K
Crystal grow
*PLUS
pH: 7.5
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-ID
17-10 mg/mlenzyme1drop
21 mMEDTA1drop
315 mM2-mercaptoethanol1drop
450-100 mMpotassium chloride1drop
520-50 mMpotassium phosphate1drop
648 %satammonium sulfate1reservoir
720 mM2-mercaptoethanol1reservoir
8100 mMTris-HCl1reservoir

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Data collection

DiffractionMean temperature: 95 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RU200 / Wavelength: 1.5418
DetectorType: RIGAKU RAXIS / Detector: IMAGE PLATE / Date: Nov 25, 1998
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2→6 Å / Num. all: 24936 / Num. obs: 24936 / % possible obs: 100 % / Observed criterion σ(F): 3 / Observed criterion σ(I): 1 / Redundancy: 10.9 % / Biso Wilson estimate: 7 Å2 / Rmerge(I) obs: 0.083 / Net I/σ(I): 25.79
Reflection shellResolution: 2→2.07 Å / Rmerge(I) obs: 0.214 / Num. unique all: 2568 / % possible all: 99.6
Reflection shell
*PLUS
% possible obs: 99.6 %

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Processing

Software
NameVersionClassification
AMoREphasing
CNS0.5refinement
SCALEPACKdata scaling
RefinementResolution: 2→6 Å / Rfactor Rfree error: 0.007 / Data cutoff high absF: 3556497.79 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 3 / σ(I): 1 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.225 1141 4.8 %RANDOM
Rwork0.212 ---
obs0.212 23582 94.6 %-
all-24936 --
Solvent computationSolvent model: FLAT MODEL / Bsol: 96.21 Å2 / ksol: 0.996 e/Å3
Displacement parametersBiso mean: 16.9 Å2
Baniso -1Baniso -2Baniso -3
1-0 Å20 Å20 Å2
2--0 Å20 Å2
3---0 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.26 Å0.24 Å
Luzzati d res low-5 Å
Luzzati sigma a0.13 Å0.11 Å
Refinement stepCycle: LAST / Resolution: 2→6 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2157 0 15 86 2258
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.006
X-RAY DIFFRACTIONc_bond_d_na
X-RAY DIFFRACTIONc_bond_d_prot
X-RAY DIFFRACTIONc_angle_d
X-RAY DIFFRACTIONc_angle_d_na
X-RAY DIFFRACTIONc_angle_d_prot
X-RAY DIFFRACTIONc_angle_deg1.5
X-RAY DIFFRACTIONc_angle_deg_na
X-RAY DIFFRACTIONc_angle_deg_prot
X-RAY DIFFRACTIONc_dihedral_angle_d24
X-RAY DIFFRACTIONc_dihedral_angle_d_na
X-RAY DIFFRACTIONc_dihedral_angle_d_prot
X-RAY DIFFRACTIONc_improper_angle_d0.82
X-RAY DIFFRACTIONc_improper_angle_d_na
X-RAY DIFFRACTIONc_improper_angle_d_prot
X-RAY DIFFRACTIONc_mcbond_it
X-RAY DIFFRACTIONc_mcangle_it
X-RAY DIFFRACTIONc_scbond_it
X-RAY DIFFRACTIONc_scangle_it
LS refinement shellResolution: 2→2.12 Å / Rfactor Rfree error: 0.018 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.246 194 5.2 %
Rwork0.218 3503 -
obs--89.4 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2WATER_REP.PARAMWATER.TOP
X-RAY DIFFRACTION3ION.PARAMION.TOP
Software
*PLUS
Name: CNS / Version: 0.5 / Classification: refinement
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg24
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg0.82

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