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- PDB-6pff: Crystal structure of TS-DHFR from Cryptosporidium hominis in comp... -

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Basic information

Entry
Database: PDB / ID: 6pff
TitleCrystal structure of TS-DHFR from Cryptosporidium hominis in complex with NADPH, FdUMP and 2-(4-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl)benzamido)-4-cyanobenzoic acid.
ComponentsBifunctional dihydrofolate reductase-thymidylate synthase
Keywordstransferase/transferase inhibitor / Inhibitor / TS / TS-DHFR / TRANSFERASE / transferase-transferase inhibitor complex
Function / homology
Function and homology information


thymidylate synthase activity / dTMP biosynthetic process / dihydrofolate reductase activity / tetrahydrofolate biosynthetic process / one-carbon metabolic process / methylation / nucleotide binding / mitochondrion / cytosol
Similarity search - Function
Bifunctional dihydrofolate reductase/thymidylate synthase / Thymidylate Synthase; Chain A / Thymidylate synthase/dCMP hydroxymethylase domain / Thymidylate synthase, active site / Thymidylate synthase active site. / Thymidylate synthase / Thymidylate synthase/dCMP hydroxymethylase / 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, active site / Thymidylate synthase active site. / Thymidylate synthase / Thymidylate synthase/dCMP hydroxymethylase / 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 (DHFR) domain profile. / Dihydrofolate reductase domain / Dihydrofolate reductase / Dihydrofolate reductase-like domain superfamily / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
METHOTREXATE / Chem-NDP / Chem-OG1 / 5-FLUORO-2'-DEOXYURIDINE-5'-MONOPHOSPHATE / Bifunctional dihydrofolate reductase-thymidylate synthase
Similarity search - Component
Biological speciesCryptosporidium hominis (eukaryote)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.982 Å
AuthorsCzyzyk, D.J. / Valhondo, M. / Jorgensen, W.L. / Anderson, K.S.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI083146 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)5T32AI007404 United States
CitationJournal: Eur.J.Med.Chem. / Year: 2019
Title: Structure activity relationship towards design of cryptosporidium specific thymidylate synthase inhibitors.
Authors: Czyzyk, D.J. / Valhondo, M. / Deiana, L. / Tirado-Rives, J. / Jorgensen, W.L. / Anderson, K.S.
History
DepositionJun 21, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 2, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
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
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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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
C: Bifunctional dihydrofolate reductase-thymidylate synthase
D: Bifunctional dihydrofolate reductase-thymidylate synthase
E: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)311,56530
Polymers301,3135
Non-polymers10,25225
Water25214
1
A: Bifunctional dihydrofolate reductase-thymidylate synthase
B: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,62612
Polymers120,5252
Non-polymers4,10110
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14500 Å2
ΔGint-75 kcal/mol
Surface area37340 Å2
MethodPISA
2
C: Bifunctional dihydrofolate reductase-thymidylate synthase
D: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,62612
Polymers120,5252
Non-polymers4,10110
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14580 Å2
ΔGint-78 kcal/mol
Surface area37110 Å2
MethodPISA
3
E: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules

E: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,62612
Polymers120,5252
Non-polymers4,10110
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_455-x-1,y,-z1
Buried area14460 Å2
ΔGint-75 kcal/mol
Surface area36720 Å2
MethodPISA
Unit cell
Length a, b, c (Å)213.424, 116.794, 221.760
Angle α, β, γ (deg.)90.00, 95.69, 90.00
Int Tables number5
Space group name H-MC121

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Components

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Protein , 1 types, 5 molecules ABCDE

#1: Protein
Bifunctional dihydrofolate reductase-thymidylate synthase


Mass: 60262.520 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cryptosporidium hominis (eukaryote) / Gene: CHUDEA4_4460 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): PA-414 / References: UniProt: A0A0S4TER9

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Non-polymers , 6 types, 39 molecules

#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-OG1 / 2-({4-[(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl]benzene-1-carbonyl}amino)-4-cyanobenzoic acid


Mass: 428.400 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C22H16N6O4 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-MTX / METHOTREXATE


Mass: 454.439 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C20H22N8O5
#6: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: SO4
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 14 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: 4.69 Å3/Da / Density % sol: 73.8 %
Crystal growTemperature: 295.15 K / Method: vapor diffusion, hanging drop / pH: 8
Details: Well solution 18 % PEG 6000, 0.2 M ammonium sulfate, 0.06 M lithium sulfate, 0.1 M Tris Drop ratio 2:1 enzyme mix/well solution

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-E / Wavelength: 0.97918 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Aug 16, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97918 Å / Relative weight: 1
ReflectionResolution: 2.98→50 Å / Num. obs: 108730 / % possible obs: 98.2 % / Redundancy: 3.7 % / CC1/2: 0.993 / Rsym value: 0.128 / Net I/σ(I): 9.62
Reflection shellResolution: 2.98→3.16 Å / Redundancy: 3.6 % / Mean I/σ(I) obs: 1.9 / Num. unique obs: 15852 / CC1/2: 0.829 / Rsym value: 0.659 / % possible all: 89.3

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Processing

Software
NameVersionClassification
PHENIX(1.15.2_3472: 000)refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4Q0E

4q0e


Resolution: 2.982→48.817 Å / SU ML: 0.42 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 26.27
RfactorNum. reflection% reflection
Rfree0.2393 5431 5 %
Rwork0.2085 --
obs0.21 108621 98.12 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.982→48.817 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms20215 0 695 14 20924
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00221429
X-RAY DIFFRACTIONf_angle_d0.53429152
X-RAY DIFFRACTIONf_dihedral_angle_d9.35512424
X-RAY DIFFRACTIONf_chiral_restr0.0443117
X-RAY DIFFRACTIONf_plane_restr0.0033717
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.9815-3.01540.5458880.42471681X-RAY DIFFRACTION48
3.0154-3.05090.34511840.34643471X-RAY DIFFRACTION99
3.0509-3.08810.38171780.30913420X-RAY DIFFRACTION100
3.0881-3.12720.39381860.31843526X-RAY DIFFRACTION100
3.1272-3.16830.31331830.30223459X-RAY DIFFRACTION100
3.1683-3.21170.34171850.28923527X-RAY DIFFRACTION100
3.2117-3.25760.3111820.2613446X-RAY DIFFRACTION100
3.2576-3.30620.28241830.26733486X-RAY DIFFRACTION100
3.3062-3.35780.31941850.27723498X-RAY DIFFRACTION100
3.3578-3.41290.3021810.27323440X-RAY DIFFRACTION100
3.4129-3.47170.30911870.26573550X-RAY DIFFRACTION100
3.4717-3.53480.24941820.24463463X-RAY DIFFRACTION100
3.5348-3.60280.26521830.22743470X-RAY DIFFRACTION100
3.6028-3.67630.2771840.24253497X-RAY DIFFRACTION100
3.6763-3.75620.27991830.2293481X-RAY DIFFRACTION100
3.7562-3.84360.241840.21673490X-RAY DIFFRACTION100
3.8436-3.93960.25261860.21493521X-RAY DIFFRACTION100
3.9396-4.04610.2581830.20963487X-RAY DIFFRACTION100
4.0461-4.16510.20171840.1973496X-RAY DIFFRACTION100
4.1651-4.29950.22971840.18443488X-RAY DIFFRACTION100
4.2995-4.4530.20461850.16743514X-RAY DIFFRACTION100
4.453-4.63120.19331830.16313500X-RAY DIFFRACTION100
4.6312-4.84180.20961850.15813511X-RAY DIFFRACTION100
4.8418-5.09680.18521850.16263507X-RAY DIFFRACTION100
5.0968-5.41570.18361830.17863492X-RAY DIFFRACTION100
5.4157-5.83330.22841870.19373550X-RAY DIFFRACTION100
5.8333-6.41910.21681860.19523524X-RAY DIFFRACTION100
6.4191-7.34530.17771850.17783526X-RAY DIFFRACTION100
7.3453-9.2440.18831880.15283560X-RAY DIFFRACTION100
9.244-48.82370.19621890.18093609X-RAY DIFFRACTION99

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