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- PDB-4hej: Discovery of Selective and Potent Inhibitors of Gram-positive Bac... -

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Basic information

Entry
Database: PDB / ID: 4hej
TitleDiscovery of Selective and Potent Inhibitors of Gram-positive Bacterial Thymidylate Kinase (TMK): Compund 16
ComponentsThymidylate kinase
KeywordsTRANSFERASE/TRANSFERASE INHIBITOR / TMK / inhibitor / TMP / antibacterial / DNA repair / kinase / dTMP ATP / phosphorylation / TRANSFERASE-TRANSFERASE INHIBITOR complex
Function / homology
Function and homology information


dTMP kinase / thymidylate kinase activity / dTDP biosynthetic process / dTTP biosynthetic process / phosphorylation / ATP binding
Similarity search - Function
Thymidylate kinase, conserved site / Thymidylate kinase signature. / Thymidylate kinase / Thymidylate kinase-like domain / Thymidylate kinase / P-loop containing nucleotide triphosphate hydrolases / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Chem-14D / Thymidylate kinase
Similarity search - Component
Biological speciesStaphylococcus aureus subsp. aureus (bacteria)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsMartinez-Botella, G. / Breen, J. / Duffy, J. / Dumas, J. / Geng, B. / Gowers, I. / Green, O. / Guler, S. / Hentemann, M. / Hernandez-Juan, F. ...Martinez-Botella, G. / Breen, J. / Duffy, J. / Dumas, J. / Geng, B. / Gowers, I. / Green, O. / Guler, S. / Hentemann, M. / Hernandez-Juan, F. / Joseph-McCarthy, D. / Kawatkar, S. / Larsen, N. / Lazari, O. / Loch, J. / Macritchie, J.
CitationJournal: J.Med.Chem. / Year: 2012
Title: Discovery of Selective and Potent Inhibitors of Gram-Positive Bacterial Thymidylate Kinase (TMK).
Authors: Martinez-Botella, G. / Breen, J.N. / Duffy, J.E. / Dumas, J. / Geng, B. / Gowers, I.K. / Green, O.M. / Guler, S. / Hentemann, M.F. / Hernandez-Juan, F.A. / Joseph-McCarthy, D. / Kawatkar, S. ...Authors: Martinez-Botella, G. / Breen, J.N. / Duffy, J.E. / Dumas, J. / Geng, B. / Gowers, I.K. / Green, O.M. / Guler, S. / Hentemann, M.F. / Hernandez-Juan, F.A. / Joseph-McCarthy, D. / Kawatkar, S. / Larsen, N.A. / Lazari, O. / Loch, J.T. / Macritchie, J.A. / McKenzie, A.R. / Newman, J.V. / Olivier, N.B. / Otterson, L.G. / Owens, A.P. / Read, J. / Sheppard, D.W. / Keating, T.A.
History
DepositionOct 3, 2012Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 24, 2012Provider: repository / Type: Initial release
Revision 1.1Jan 2, 2013Group: Database references
Revision 1.2Feb 28, 2024Group: 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 / software / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _software.name / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Thymidylate kinase
B: Thymidylate kinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)47,3693
Polymers46,9092
Non-polymers4591
Water3,477193
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1980 Å2
ΔGint-18 kcal/mol
Surface area18280 Å2
MethodPISA
Unit cell
Length a, b, c (Å)45.348, 90.308, 50.070
Angle α, β, γ (deg.)90.00, 100.69, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Thymidylate kinase / / dTMP kinase


Mass: 23454.586 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus subsp. aureus (bacteria)
Strain: N315 / Gene: SA0440, tmk / Plasmid: pET30 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21-Codon + / References: UniProt: P65249, dTMP kinase
#2: Chemical ChemComp-14D / 5-methyl-1-[(3S)-1-{3-[3-(trifluoromethyl)phenoxy]benzyl}piperidin-3-yl]pyrimidine-2,4(1H,3H)-dione


Mass: 459.461 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H24F3N3O3
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 193 / 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: 2.15 Å3/Da / Density % sol: 42.73 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 6.8
Details: To obtain the inhibitor bound crystal form of TMK-S.aureus crystals were initially grown in the absence of compound using the sitting drop method at 20 C with a reservoir solution of 100 mM ...Details: To obtain the inhibitor bound crystal form of TMK-S.aureus crystals were initially grown in the absence of compound using the sitting drop method at 20 C with a reservoir solution of 100 mM PCPT (propionate-cacodylate-bistris propane buffer) pH 7-8, 21-24% PEG 3350, 200 mM Mg2Cl using 1:1 protein:reservoir solution with the protein solution at 13 mg/mL. Crystals were harvested and soaked overnight in a solution containing 100 mM PCPT, 35% PEG 3350, 200 mM Mg2Cl and 1-2 mM TK-666 from a 100 mM DMSO stock. After soaking the crystals were cryoprotected by soaking for 15 minutes in compound-soak solution supplemented with 20% ethylene glycol. Cryoprotected crystals were mounted on nylon loops and flash-cooled in liquid nitrogen., VAPOR DIFFUSION, SITTING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 140 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU FR-E+ SUPERBRIGHT / Wavelength: 1.54 Å
DetectorType: RIGAKU SATURN 944+ / Detector: CCD / Date: Aug 29, 2008 / Details: microfocus rotating anode with 70 uM focal spot
RadiationMonochromator: Varimax confocal X-ray optical assembly / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54 Å / Relative weight: 1
ReflectionResolution: 2.1→25.68 Å / Num. all: 26805 / Num. obs: 20571 / % possible obs: 76.7 % / Observed criterion σ(F): 1 / Observed criterion σ(I): 1.3 / Redundancy: 3.38 % / Biso Wilson estimate: 28.73 Å2 / Rmerge(I) obs: 0.075 / Net I/σ(I): 11.4
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique allDiffraction-ID% possible all
2-2.071.750.1473.32669123.5
2.07-2.152.150.1154.12679136.7
2.15-2.252.570.1634.32658149.9
2.25-2.372.890.1085.62688169
2.37-2.523.40.0946.92689194.8
2.52-2.713.740.0798.12636198

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Processing

Software
NameVersionClassification
JDirectordata collection
AMoREphasing
BUSTER2.11.2refinement
d*TREKdata reduction
d*TREKdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2→25.68 Å / Cor.coef. Fo:Fc: 0.9336 / Cor.coef. Fo:Fc free: 0.9059 / SU R Cruickshank DPI: 0.318 / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.2347 987 5.03 %RANDOM, 5%
Rwork0.1929 ---
obs0.195 19634 73.24 %-
Displacement parametersBiso mean: 30.16 Å2
Baniso -1Baniso -2Baniso -3
1-0.0116 Å20 Å2-4.3585 Å2
2--2.7813 Å20 Å2
3----2.7929 Å2
Refine analyzeLuzzati coordinate error obs: 0.245 Å
Refinement stepCycle: LAST / Resolution: 2→25.68 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3147 0 33 193 3373
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.013243HARMONIC2
X-RAY DIFFRACTIONt_angle_deg1.154382HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d1162SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes95HARMONIC2
X-RAY DIFFRACTIONt_gen_planes470HARMONIC5
X-RAY DIFFRACTIONt_it3243HARMONIC20
X-RAY DIFFRACTIONt_omega_torsion3.01
X-RAY DIFFRACTIONt_other_torsion18.26
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_chiral_improper_torsion423SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies1HARMONIC1
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact3967SEMIHARMONIC4
LS refinement shellResolution: 2→2.11 Å / Total num. of bins used: 10
RfactorNum. reflection% reflection
Rfree0.1143 4 3.25 %
Rwork0.219 119 -
all0.2149 123 -
obs--73.24 %

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