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

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Basic information

Entry
Database: PDB / ID: 6pfh
TitleCrystal structure of TS-DHFR from Cryptosporidium hominis in complex with NADPH, FdUMP and 2-(2-(4-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl)benzamido)-4-cyanophenyl)acetic 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-OFD / 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: 2.94 Å
AuthorsCzyzyk, D.C. / 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)311,15525
Polymers301,3135
Non-polymers9,84220
Water00
1
A: Bifunctional dihydrofolate reductase-thymidylate synthase
B: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,46210
Polymers120,5252
Non-polymers3,9378
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14170 Å2
ΔGint-50 kcal/mol
Surface area37940 Å2
MethodPISA
2
C: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules

C: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,46210
Polymers120,5252
Non-polymers3,9378
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_558-x,y,-z+31
Buried area14100 Å2
ΔGint-51 kcal/mol
Surface area37220 Å2
MethodPISA
3
D: Bifunctional dihydrofolate reductase-thymidylate synthase
E: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,46210
Polymers120,5252
Non-polymers3,9378
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14120 Å2
ΔGint-47 kcal/mol
Surface area37850 Å2
MethodPISA
Unit cell
Length a, b, c (Å)214.110, 116.936, 221.376
Angle α, β, γ (deg.)90.00, 95.43, 90.00
Int Tables number5
Space group name H-MC121

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Components

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


Mass: 442.427 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C23H18N6O4 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-MTX / METHOTREXATE


Mass: 454.439 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C20H22N8O5
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: 4.71 Å3/Da / Density % sol: 73.9 %
Crystal growTemperature: 295.15 K / Method: vapor diffusion, hanging drop / pH: 8
Details: Well solution 20 % 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: NSLS-II / Beamline: 17-ID-2 / Wavelength: 0.979303 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Jun 7, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979303 Å / Relative weight: 1
ReflectionResolution: 2.94→30 Å / Num. obs: 115109 / % possible obs: 99.3 % / Redundancy: 7 % / CC1/2: 0.996 / Rsym value: 0.152 / Net I/σ(I): 10.51
Reflection shellResolution: 2.94→3.12 Å / Redundancy: 7 % / Mean I/σ(I) obs: 1.76 / Num. unique obs: 17791 / CC1/2: 0.791 / Rsym value: 0.93 / % possible all: 96.2

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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: 2.94→29.719 Å / SU ML: 0.37 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 24.44
RfactorNum. reflection% reflection
Rfree0.2191 5752 5 %
Rwork0.1884 --
obs0.1899 115049 99.33 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.94→29.719 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms20293 0 675 0 20968
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00221493
X-RAY DIFFRACTIONf_angle_d0.50429219
X-RAY DIFFRACTIONf_dihedral_angle_d9.47612492
X-RAY DIFFRACTIONf_chiral_restr0.0433122
X-RAY DIFFRACTIONf_plane_restr0.0033724
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.9389-2.97220.37331580.3522991X-RAY DIFFRACTION82
2.9722-3.00720.3371910.29773620X-RAY DIFFRACTION100
3.0072-3.04380.3331910.293632X-RAY DIFFRACTION100
3.0438-3.08230.34361920.27413649X-RAY DIFFRACTION100
3.0823-3.12280.3221930.29963657X-RAY DIFFRACTION100
3.1228-3.16550.3611910.29073636X-RAY DIFFRACTION100
3.1655-3.21070.35171940.27683682X-RAY DIFFRACTION100
3.2107-3.25860.28791900.24583619X-RAY DIFFRACTION100
3.2586-3.30940.31421920.24523643X-RAY DIFFRACTION100
3.3094-3.36360.29511910.24873635X-RAY DIFFRACTION100
3.3636-3.42150.28781920.24413645X-RAY DIFFRACTION100
3.4215-3.48360.2671940.24133701X-RAY DIFFRACTION100
3.4836-3.55050.23031910.213629X-RAY DIFFRACTION100
3.5505-3.62290.23261930.19783651X-RAY DIFFRACTION100
3.6229-3.70150.23631920.20553670X-RAY DIFFRACTION100
3.7015-3.78750.22711910.19023627X-RAY DIFFRACTION100
3.7875-3.8820.23241940.18653680X-RAY DIFFRACTION100
3.882-3.98670.19321910.17973639X-RAY DIFFRACTION100
3.9867-4.10370.21451930.17343669X-RAY DIFFRACTION100
4.1037-4.23580.19691930.15193656X-RAY DIFFRACTION100
4.2358-4.38680.16071940.14553683X-RAY DIFFRACTION100
4.3868-4.56180.18321920.14463654X-RAY DIFFRACTION100
4.5618-4.76860.16361940.14493669X-RAY DIFFRACTION100
4.7686-5.01890.16521940.14443682X-RAY DIFFRACTION100
5.0189-5.33170.18241940.16483699X-RAY DIFFRACTION100
5.3317-5.74060.23431930.1753659X-RAY DIFFRACTION100
5.7406-6.31330.20921940.18933699X-RAY DIFFRACTION100
6.3133-7.21540.20111960.17333708X-RAY DIFFRACTION100
7.2154-9.04790.16441960.15523721X-RAY DIFFRACTION100
9.0479-29.72010.18211980.1713792X-RAY DIFFRACTION100

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