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- PDB-4ky8: Crystal structure of TS-DHFR from Cryptosporidium hominis in comp... -

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Basic information

Entry
Database: PDB / ID: 4ky8
TitleCrystal structure of TS-DHFR from Cryptosporidium hominis in complex with NADPH, methotrexate, FdUMP and 4-((2-amino-6-methyl-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)thio)-2-chlorophenyl)-L-glutamic acid
ComponentsBifunctional thymidylate synthase-dihydrofolate reductase
KeywordsTRANSFERASE / OXIDOREDUCTASE/INHIBITOR / bifunctional enzyme / oxidoreductase / OXIDOREDUCTASE-INHIBITOR complex
Function / homology
Function and homology information


Thymidylate Synthase; Chain A / Thymidylate synthase/dCMP hydroxymethylase domain / Dihydrofolate Reductase, subunit A / Dihydrofolate Reductase, subunit A / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Chem-1UF / METHOTREXATE / Chem-NDP / 5-FLUORO-2'-DEOXYURIDINE-5'-MONOPHOSPHATE / :
Similarity search - Component
Biological speciesCryptosporidium hominis (eukaryote)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.084 Å
AuthorsKumar, V.P. / Anderson, K.S.
CitationJournal: Bioorg.Med.Chem.Lett. / Year: 2013
Title: Substituted pyrrolo[2,3-d]pyrimidines as Cryptosporidium hominis thymidylate synthase inhibitors.
Authors: Kumar, V.P. / Frey, K.M. / Wang, Y. / Jain, H.K. / Gangjee, A. / Anderson, K.S.
History
DepositionMay 28, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 21, 2013Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2013Group: Database references
Revision 1.2Nov 15, 2017Group: Refinement description / Category: software
Revision 1.3Sep 20, 2023Group: 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 / pdbx_initial_refinement_model / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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: Bifunctional thymidylate synthase-dihydrofolate reductase
B: Bifunctional thymidylate synthase-dihydrofolate reductase
C: Bifunctional thymidylate synthase-dihydrofolate reductase
D: Bifunctional thymidylate synthase-dihydrofolate reductase
E: Bifunctional thymidylate synthase-dihydrofolate reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)311,34225
Polymers301,3135
Non-polymers10,03020
Water00
1
A: Bifunctional thymidylate synthase-dihydrofolate reductase
B: Bifunctional thymidylate synthase-dihydrofolate reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,53710
Polymers120,5252
Non-polymers4,0128
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8340 Å2
ΔGint-26 kcal/mol
Surface area40190 Å2
MethodPISA
2
C: Bifunctional thymidylate synthase-dihydrofolate reductase
D: Bifunctional thymidylate synthase-dihydrofolate reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,53710
Polymers120,5252
Non-polymers4,0128
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8380 Å2
ΔGint-28 kcal/mol
Surface area40360 Å2
MethodPISA
3
E: Bifunctional thymidylate synthase-dihydrofolate reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)62,2685
Polymers60,2631
Non-polymers2,0064
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)214.162, 115.665, 218.767
Angle α, β, γ (deg.)90.00, 94.36, 90.00
Int Tables number5
Space group name H-MC121

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Components

#1: Protein
Bifunctional thymidylate synthase-dihydrofolate reductase


Mass: 60262.520 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cryptosporidium hominis (eukaryote) / Gene: Chro.40506, TS-DHFR / Plasmid: pTrc99A / Production host: Escherichia coli (E. coli) / Strain (production host): PA414
References: UniProt: Q5CGA3, thymidylate synthase, dihydrofolate reductase
#2: Chemical
ChemComp-NDP / NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE


Mass: 745.421 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C21H30N7O17P3
#3: Chemical
ChemComp-UFP / 5-FLUORO-2'-DEOXYURIDINE-5'-MONOPHOSPHATE


Type: DNA linking / Mass: 326.172 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C9H12FN2O8P
#4: Chemical
ChemComp-MTX / METHOTREXATE


Mass: 454.439 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C20H22N8O5
#5: Chemical
ChemComp-1UF / N-{4-[(2-amino-6-methyl-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)sulfanyl]-2-chlorobenzoyl}-L-glutamic acid


Mass: 479.894 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C19H18ClN5O6S

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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: 4.48 Å3/Da / Density % sol: 72.56 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 14% (w/v) PEG 6000, 60 mM ammonium sulfate, 200 mM lithium sulfate and 100 mM Tris pH 8.0. ChTS-DHFR enzyme (~ 7 mg/ml) with 1 mM NADPH, 1 mM MTX, 1 mM FdUMP and 0.5 mM inhibitor., VAPOR ...Details: 14% (w/v) PEG 6000, 60 mM ammonium sulfate, 200 mM lithium sulfate and 100 mM Tris pH 8.0. ChTS-DHFR enzyme (~ 7 mg/ml) with 1 mM NADPH, 1 mM MTX, 1 mM FdUMP and 0.5 mM inhibitor., VAPOR DIFFUSION, HANGING DROP, temperature 298K

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Data collection

DiffractionMean temperature: 77 K
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X25 / Wavelength: 1.1 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Apr 16, 2013 / Details: monochromator
RadiationMonochromator: Double silicon (111) crystal monochromator with cryogenically-cooled first crystal and sagittally-bent second crystal horizontally focusing at 3.3:1 demagnification
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.1 Å / Relative weight: 1
ReflectionResolution: 3.08→48.12 Å / Num. obs: 70173 / % possible obs: 71.6 % / Observed criterion σ(F): 1.5 / Redundancy: 3 % / Biso Wilson estimate: 70.12 Å2 / Rmerge(I) obs: 0.207 / Rsym value: 0.207 / Net I/σ(I): 4.1
Reflection shell
Resolution (Å)Redundancy (%)Diffraction-ID% possible all
3.1-3.151.3111.3
3.15-3.211.5115.6
3.21-3.271.7121.6
3.27-3.342128.3
3.34-3.412.1135.3
3.41-3.492.2144.8
3.49-3.582.3153.3
3.58-3.682.4161.6
3.68-3.782.5172.3
3.78-3.912.7189.6
3.91-4.043197.1
4.04-4.213.1199.4
4.21-4.43.3199.8
4.4-4.633.4199.9
4.63-4.923.3199.9
4.92-5.33.51100
5.3-5.833.3199.9
5.83-6.673.5199.9
6.67-8.43.4199.9
8.4-503.3199.8

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
SCALEPACKdata scaling
PHENIX1.8.1_1168refinement
PDB_EXTRACT3.11data extraction
CBASSdata collection
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1QZF

1qzf


Resolution: 3.084→48.12 Å / Occupancy max: 1 / Occupancy min: 1 / SU ML: 0.39 / σ(F): 1.34 / Phase error: 29.7 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2663 1959 2.8 %
Rwork0.232 --
obs0.233 70048 71.2 %
all-70048 -
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 82.3969 Å2
Refinement stepCycle: LAST / Resolution: 3.084→48.12 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms20570 0 670 0 21240
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00221760
X-RAY DIFFRACTIONf_angle_d0.54329505
X-RAY DIFFRACTIONf_dihedral_angle_d12.1258190
X-RAY DIFFRACTIONf_chiral_restr0.0393115
X-RAY DIFFRACTIONf_plane_restr0.0023755
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.084-3.16110.4148220.3719736X-RAY DIFFRACTION11
3.1611-3.24650.3879390.37491260X-RAY DIFFRACTION19
3.2465-3.3420.369530.34961901X-RAY DIFFRACTION28
3.342-3.44990.3566700.3422694X-RAY DIFFRACTION40
3.4499-3.57310.29891030.30323586X-RAY DIFFRACTION53
3.5731-3.71610.34141270.29274331X-RAY DIFFRACTION64
3.7161-3.88520.28521820.27985768X-RAY DIFFRACTION85
3.8852-4.08990.29461850.25986633X-RAY DIFFRACTION97
4.0899-4.3460.26621950.21146844X-RAY DIFFRACTION100
4.346-4.68130.24041840.19086828X-RAY DIFFRACTION100
4.6813-5.15190.2221990.19126790X-RAY DIFFRACTION100
5.1519-5.89630.24771980.21276851X-RAY DIFFRACTION100
5.8963-7.42430.251930.23586878X-RAY DIFFRACTION100
7.4243-48.12580.25312090.21036989X-RAY DIFFRACTION100

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