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

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Basic information

Entry
Database: PDB / ID: 6pfd
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)terephthalic 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-OEJ / 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: 3.324 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)311,18025
Polymers301,3135
Non-polymers9,86720
Water00
1
A: Bifunctional dihydrofolate reductase-thymidylate synthase
B: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,47210
Polymers120,5252
Non-polymers3,9478
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14160 Å2
ΔGint-49 kcal/mol
Surface area37290 Å2
MethodPISA
2
C: Bifunctional dihydrofolate reductase-thymidylate synthase
D: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,47210
Polymers120,5252
Non-polymers3,9478
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14070 Å2
ΔGint-47 kcal/mol
Surface area37130 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)124,47210
Polymers120,5252
Non-polymers3,9478
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_858-x+3,y,-z+31
Buried area14030 Å2
ΔGint-46 kcal/mol
Surface area36630 Å2
MethodPISA
Unit cell
Length a, b, c (Å)213.033, 115.887, 220.808
Angle α, β, γ (deg.)90.00, 95.05, 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 / Comment: inhibitor*YM
#4: Chemical
ChemComp-OEJ / 2-({4-[(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl]benzene-1-carbonyl}amino)benzene-1,4-dicarboxylic acid


Mass: 447.400 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C22H17N5O6 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-MTX / METHOTREXATE


Mass: 454.439 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C20H22N8O5 / Comment: chemotherapy*YM
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: 4.63 Å3/Da / Density % sol: 73.5 %
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: 3.32→50 Å / Num. obs: 75886 / % possible obs: 95.9 % / Redundancy: 3.7 % / CC1/2: 0.987 / Rsym value: 0.163 / Net I/σ(I): 9.04
Reflection shellResolution: 3.32→3.52 Å / Redundancy: 3.1 % / Mean I/σ(I) obs: 1.86 / Num. unique obs: 10354 / CC1/2: 0.816 / Rsym value: 0.714 / % possible all: 81.6

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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: 3.324→48.426 Å / SU ML: 0.48 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 27.92
RfactorNum. reflection% reflection
Rfree0.2615 3786 5 %
Rwork0.2197 --
obs0.2217 75746 95.81 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 3.324→48.426 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms20115 0 675 0 20790
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00221314
X-RAY DIFFRACTIONf_angle_d0.50529018
X-RAY DIFFRACTIONf_dihedral_angle_d8.83512341
X-RAY DIFFRACTIONf_chiral_restr0.0433114
X-RAY DIFFRACTIONf_plane_restr0.0033711
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.3243-3.36640.3615600.31561159X-RAY DIFFRACTION41
3.3664-3.41060.3691280.39032424X-RAY DIFFRACTION89
3.4106-3.45740.52841330.41522535X-RAY DIFFRACTION90
3.4574-3.50670.42941410.3482689X-RAY DIFFRACTION99
3.5067-3.55910.30841460.28022763X-RAY DIFFRACTION99
3.5591-3.61470.34981430.30772714X-RAY DIFFRACTION99
3.6147-3.67390.51531400.44642646X-RAY DIFFRACTION95
3.6739-3.73720.441410.35792684X-RAY DIFFRACTION98
3.7372-3.80520.31491450.25842757X-RAY DIFFRACTION99
3.8052-3.87830.39161410.31892664X-RAY DIFFRACTION97
3.8783-3.95740.43791330.37482578X-RAY DIFFRACTION93
3.9574-4.04350.2421450.24212760X-RAY DIFFRACTION99
4.0435-4.13750.26941450.21342740X-RAY DIFFRACTION99
4.1375-4.24090.25591440.19842741X-RAY DIFFRACTION99
4.2409-4.35550.24391450.17942752X-RAY DIFFRACTION99
4.3555-4.48350.22241450.16642756X-RAY DIFFRACTION99
4.4835-4.62820.19061470.16272787X-RAY DIFFRACTION99
4.6282-4.79340.22041430.15842725X-RAY DIFFRACTION99
4.7934-4.98520.20581460.15772773X-RAY DIFFRACTION99
4.9852-5.21180.20231450.16832753X-RAY DIFFRACTION99
5.2118-5.48620.21231470.18372786X-RAY DIFFRACTION99
5.4862-5.82940.20671460.18542774X-RAY DIFFRACTION99
5.8294-6.27860.21571450.18172760X-RAY DIFFRACTION99
6.2786-6.90890.21931460.18312778X-RAY DIFFRACTION99
6.9089-7.90480.21811480.16572802X-RAY DIFFRACTION99
7.9048-9.94510.141470.14062807X-RAY DIFFRACTION99
9.9451-48.43080.20151510.18382853X-RAY DIFFRACTION99

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