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- PDB-6n1s: Toxoplasma gondii TS-DHFR in complex with selective inhibitor 29 -

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Basic information

Entry
Database: PDB / ID: 6n1s
TitleToxoplasma gondii TS-DHFR in complex with selective inhibitor 29
ComponentsBifunctional dihydrofolate reductase-thymidylate synthase
Keywordsoxidoreductase/oxidoreductase inhibitor / TOXOPLASMA GONDII / DHFR / OXIDOREDUCTASE / oxidoreductase-oxidoreductase inhibitor complex
Function / homology
Function and homology information


thymidylate synthase / thymidylate synthase activity / dTMP biosynthetic process / dihydrofolate reductase / dihydrofolate reductase activity / tetrahydrofolate biosynthetic process / one-carbon metabolic process / methylation
Similarity search - Function
Bifunctional dihydrofolate reductase/thymidylate synthase / 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 ...Bifunctional dihydrofolate reductase/thymidylate synthase / 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 / Dihydrofolate Reductase, subunit A / Dihydrofolate Reductase, subunit A / Dihydrofolate reductase conserved site / Dihydrofolate reductase (DHFR) domain signature. / Dihydrofolate reductase domain / Dihydrofolate reductase / Dihydrofolate reductase (DHFR) domain profile. / Dihydrofolate reductase-like domain superfamily / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
10-PROPARGYL-5,8-DIDEAZAFOLIC ACID / Chem-K9A / Chem-NDP / 2'-DEOXYURIDINE 5'-MONOPHOSPHATE / Bifunctional dihydrofolate reductase-thymidylate synthase
Similarity search - Component
Biological speciesToxoplasma gondii (eukaryote)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 3 Å
AuthorsHopper, A.T. / Brockman, A. / Wise, A. / Gould, J. / Barks, J. / Radke, J.B. / Sibley, L.D. / Zou, Y. / Thomas, S.B.
CitationJournal: J. Med. Chem. / Year: 2019
Title: Discovery of Selective Toxoplasma gondii Dihydrofolate Reductase Inhibitors for the Treatment of Toxoplasmosis.
Authors: Hopper, A.T. / Brockman, A. / Wise, A. / Gould, J. / Barks, J. / Radke, J.B. / Sibley, L.D. / Zou, Y. / Thomas, S.
History
DepositionNov 11, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 23, 2019Provider: repository / Type: Initial release
Revision 1.1Apr 24, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Oct 11, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Bifunctional dihydrofolate reductase-thymidylate synthase
B: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)132,27811
Polymers128,4292
Non-polymers3,8499
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12520 Å2
ΔGint-57 kcal/mol
Surface area39410 Å2
MethodPISA
Unit cell
Length a, b, c (Å)53.689, 145.522, 175.922
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

NCS domain segments:

Component-ID: 0 / Ens-ID: 1 / Beg auth comp-ID: PRO / Beg label comp-ID: PRO / End auth comp-ID: VAL / End label comp-ID: VAL / Refine code: 0 / Auth seq-ID: 4 - 610 / Label seq-ID: 4 - 566

Dom-IDAuth asym-IDLabel asym-ID
1AA
2BB

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Bifunctional dihydrofolate reductase-thymidylate synthase / DHFR-TS


Mass: 64214.312 Da / Num. of mol.: 2 / Fragment: DHFR-TS / Mutation: delta 49-73, delta 201-219
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Toxoplasma gondii (eukaryote) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
References: UniProt: Q07422, dihydrofolate reductase, thymidylate synthase

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Non-polymers , 5 types, 9 molecules

#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-NDP / NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Nicotinamide adenine dinucleotide phosphate


Mass: 745.421 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H30N7O17P3
#4: Chemical ChemComp-CB3 / 10-PROPARGYL-5,8-DIDEAZAFOLIC ACID


Mass: 477.469 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C24H23N5O6
#5: Chemical ChemComp-K9A / 5-{4-[3-(2-methylpyrimidin-5-yl)phenyl]piperazin-1-yl}pyrimidine-2,4-diamine


Mass: 362.432 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C19H22N8
#6: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL / Ethylene glycol


Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O2

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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: 2.68 Å3/Da / Density % sol: 54.03 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7.3
Details: Compound 29, NADPH, PDDF, and dUMP were combined with protein and then mixed with 0.1 M potassium formate buffer with 16% PEG3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL17U / Wavelength: 0.97915 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Jul 4, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97915 Å / Relative weight: 1
ReflectionResolution: 3→50 Å / Num. obs: 25031 / % possible obs: 87.8 % / Redundancy: 4.9 % / Rmerge(I) obs: 0.147 / Rpim(I) all: 0.072 / Rrim(I) all: 0.165 / Χ2: 1.205 / Net I/σ(I): 7.1
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
3-3.114.80.45225080.7650.2280.511.11390.7
3.11-3.234.80.34425320.8730.1720.3871.12590.2
3.23-3.384.80.27725490.9140.1370.3111.23890.2
3.38-3.564.80.21125230.9390.1040.2371.2989.9
3.56-3.784.80.16825080.9590.0820.1881.25589.5
3.78-4.074.90.15125070.9660.0730.1691.35588.7
4.07-4.484.90.11825300.9770.0570.1321.36188.2
4.48-5.134.80.10224520.9840.0490.1141.20887
5.13-6.464.90.09924780.9870.0470.111.04284.9
6.46-505.10.08624440.9830.0430.0971.06579.4

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
REFMACrefinement
HKL-2000data scaling
PHASERphasing
PDB_EXTRACT3.24data extraction
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4EIL

4eil


Resolution: 3→39.6 Å / Cor.coef. Fo:Fc: 0.91 / Cor.coef. Fo:Fc free: 0.876 / SU B: 38.629 / SU ML: 0.325 / SU R Cruickshank DPI: 0.4016 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R Free: 0.459
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.2415 1294 5.2 %RANDOM
Rwork0.2069 ---
obs0.2086 23728 87.71 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 158.54 Å2 / Biso mean: 44.024 Å2 / Biso min: 15.08 Å2
Baniso -1Baniso -2Baniso -3
1-1.23 Å20 Å20 Å2
2--0.73 Å20 Å2
3----1.96 Å2
Refinement stepCycle: final / Resolution: 3→39.6 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8162 0 264 0 8426
Biso mean--48.11 --
Num. residues----1020
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0138640
X-RAY DIFFRACTIONr_bond_other_d0.0120.0177950
X-RAY DIFFRACTIONr_angle_refined_deg1.3721.65711728
X-RAY DIFFRACTIONr_angle_other_deg1.5591.58318385
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.65951008
X-RAY DIFFRACTIONr_dihedral_angle_2_deg32.20721.202466
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.646151416
X-RAY DIFFRACTIONr_dihedral_angle_4_deg16.9321568
X-RAY DIFFRACTIONr_chiral_restr0.0910.21092
X-RAY DIFFRACTIONr_gen_planes_refined0.0060.029552
X-RAY DIFFRACTIONr_gen_planes_other0.0050.021904
Refine LS restraints NCS

Ens-ID: 1 / Number: 16365 / Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Rms dev position: 0.08 Å / Weight position: 0.05

Dom-IDAuth asym-ID
1A
2B
LS refinement shellResolution: 2.999→3.076 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.3 93 -
Rwork0.292 1720 -
all-1813 -
obs--88.35 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.7346-0.1150.25031.7261-1.0234.9250.00160.3318-0.1223-0.27190.11040.04170.1849-0.0989-0.1120.0457-0.01990.00940.07670.00020.220926.53598.425-76.791
22.9273-0.3351-5.93420.32420.538512.1249-0.2136-0.1030.11580.11720.3732-0.11020.29390.0746-0.15960.46340.00420.0030.5554-0.01960.30929.20155.09-67.563
30.62260.15260.5061.6254-0.66541.6773-0.0317-0.1925-0.01680.21960.05530.2002-0.1616-0.2127-0.02360.0380.02530.05410.07240.00480.307227.92282.952-38.291
41.96370.04290.23852.6849-0.87335.9503-0.02310.70820.0645-0.63860.2420.51880.147-1.0263-0.21890.213-0.0551-0.08720.47120.0380.39474.16639.422-68.052
55.6031-6.35311.07268.0751.731310.82460.4797-0.3295-0.3602-0.42270.25940.13810.774-0.1695-0.73910.2846-0.08390.00510.20740.10840.276525.53781.528-72.577
61.260.67560.56811.3401-0.06751.64210.0845-0.0063-0.1259-0.05650.0278-0.20460.06560.2137-0.11230.02790.00850.0620.0560.03430.356729.92454.375-39.316
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A3 - 252
2X-RAY DIFFRACTION2A285 - 298
3X-RAY DIFFRACTION3A310 - 610
4X-RAY DIFFRACTION4B4 - 252
5X-RAY DIFFRACTION5B285 - 300
6X-RAY DIFFRACTION6B310 - 610

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