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

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Basic information

Entry
Database: PDB / ID: 6pfi
TitleCrystal structure of TS-DHFR from Cryptosporidium hominis in complex with NADPH, FdUMP and 3-(4-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl)benzamido)-4-(carboxymethyl)benzoic 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-OEP / 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: 2.89 Å
AuthorsCzyzyk, D.J. / Anderson, K.S. / Jorgensen, W.L. / Valhondo, M.
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)311,25025
Polymers301,3135
Non-polymers9,93720
Water46826
1
A: Bifunctional dihydrofolate reductase-thymidylate synthase
B: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,50010
Polymers120,5252
Non-polymers3,9758
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14140 Å2
ΔGint-47 kcal/mol
Surface area37660 Å2
MethodPISA
2
C: Bifunctional dihydrofolate reductase-thymidylate synthase
D: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,50010
Polymers120,5252
Non-polymers3,9758
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14160 Å2
ΔGint-45 kcal/mol
Surface area37810 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)124,50010
Polymers120,5252
Non-polymers3,9758
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_455-x-1,y,-z1
Buried area14240 Å2
ΔGint-48 kcal/mol
Surface area36590 Å2
MethodPISA
Unit cell
Length a, b, c (Å)213.380, 116.741, 221.580
Angle α, β, γ (deg.)90.00, 95.05, 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 , 5 types, 46 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-OEP / 3-({4-[(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl]benzene-1-carbonyl}amino)-4-(carboxymethyl)benzoic acid


Mass: 461.427 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C23H19N5O6
#5: Chemical
ChemComp-MTX / METHOTREXATE


Mass: 454.439 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C20H22N8O5
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 26 / 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-C / Wavelength: 0.9792 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Nov 15, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9792 Å / Relative weight: 1
ReflectionResolution: 2.89→50 Å / Num. obs: 119315 / % possible obs: 98.2 % / Redundancy: 6.9 % / CC1/2: 0.985 / Rsym value: 0.255 / Net I/σ(I): 5.18
Reflection shellResolution: 2.89→3.06 Å / Redundancy: 7 % / Mean I/σ(I) obs: 1.16 / Num. unique obs: 18527 / CC1/2: 0.671 / Rsym value: 1.324 / % possible all: 95.1

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Processing

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

4q0e


Resolution: 2.89→48.589 Å / SU ML: 0.46 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 28.87 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2519 1998 1.68 %
Rwork0.2122 --
obs0.2129 119210 98.17 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.89→48.589 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms20253 0 680 26 20959
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00221458
X-RAY DIFFRACTIONf_angle_d0.50529178
X-RAY DIFFRACTIONf_dihedral_angle_d9.38312468
X-RAY DIFFRACTIONf_chiral_restr0.0433119
X-RAY DIFFRACTIONf_plane_restr0.0033721
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.8901-2.96240.37721300.36417635X-RAY DIFFRACTION90
2.9624-3.04240.36661430.33088410X-RAY DIFFRACTION99
3.0424-3.1320.35441430.30758400X-RAY DIFFRACTION99
3.132-3.2330.33331430.28498367X-RAY DIFFRACTION99
3.233-3.34860.29711440.25588451X-RAY DIFFRACTION99
3.3486-3.48260.33641430.25298391X-RAY DIFFRACTION99
3.4826-3.6410.26641410.2458255X-RAY DIFFRACTION97
3.641-3.83290.27321440.21438482X-RAY DIFFRACTION100
3.8329-4.0730.24761440.20388464X-RAY DIFFRACTION100
4.073-4.38720.23251450.17798470X-RAY DIFFRACTION99
4.3872-4.82840.20871410.1688292X-RAY DIFFRACTION97
4.8284-5.52620.19581470.17798555X-RAY DIFFRACTION100
5.5262-6.95920.25351420.20758404X-RAY DIFFRACTION98
6.9592-48.59550.20541480.18468636X-RAY DIFFRACTION99

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