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

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Basic information

Entry
Database: PDB / ID: 6pfe
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-methoxybenzoic 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-OEA / 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: 2.812 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)311,11025
Polymers301,3135
Non-polymers9,79720
Water72140
1
A: Bifunctional dihydrofolate reductase-thymidylate synthase
B: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,44410
Polymers120,5252
Non-polymers3,9198
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14050 Å2
ΔGint-51 kcal/mol
Surface area37230 Å2
MethodPISA
2
C: Bifunctional dihydrofolate reductase-thymidylate synthase
E: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,44410
Polymers120,5252
Non-polymers3,9198
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area13920 Å2
ΔGint-48 kcal/mol
Surface area37200 Å2
MethodPISA
3
D: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules

D: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,44410
Polymers120,5252
Non-polymers3,9198
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_558-x,y,-z+31
Buried area13770 Å2
ΔGint-47 kcal/mol
Surface area36970 Å2
MethodPISA
Unit cell
Length a, b, c (Å)212.859, 115.641, 219.899
Angle α, β, γ (deg.)90.00, 95.00, 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, 60 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 / Comment: inhibitor*YM
#4: Chemical
ChemComp-OEA / 2-({4-[(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl]benzene-1-carbonyl}amino)-4-methoxybenzoic acid


Mass: 433.417 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C22H19N5O5 / 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
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 40 / 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: 4.6 Å3/Da / Density % sol: 73.3 %
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: 2.8→50 Å / Num. obs: 124669 / % possible obs: 95.5 % / Redundancy: 6 % / CC1/2: 0.984 / Rsym value: 0.254 / Net I/σ(I): 5.78
Reflection shellResolution: 2.8→2.97 Å / Redundancy: 5.3 % / Mean I/σ(I) obs: 0.83 / Num. unique obs: 17614 / CC1/2: 0.5 / Rsym value: 1.629 / % possible all: 84.1

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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: 2.812→49.438 Å / SU ML: 0.47 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 31.55
RfactorNum. reflection% reflection
Rfree0.2572 1995 1.61 %
Rwork0.2269 --
obs0.2274 123995 95.95 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.812→49.438 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms20256 0 670 40 20966
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00221451
X-RAY DIFFRACTIONf_angle_d0.55229177
X-RAY DIFFRACTIONf_dihedral_angle_d10.04312480
X-RAY DIFFRACTIONf_chiral_restr0.0453122
X-RAY DIFFRACTIONf_plane_restr0.0033724
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.8122-2.88260.43651090.38616649X-RAY DIFFRACTION74
2.8826-2.96050.41261470.36818924X-RAY DIFFRACTION98
2.9605-3.04760.32291440.34338792X-RAY DIFFRACTION98
3.0476-3.1460.37621450.32468872X-RAY DIFFRACTION98
3.146-3.25840.3431430.30218784X-RAY DIFFRACTION97
3.2584-3.38880.31841410.28118618X-RAY DIFFRACTION95
3.3888-3.5430.3041470.26628959X-RAY DIFFRACTION99
3.543-3.72970.27611460.24888947X-RAY DIFFRACTION99
3.7297-3.96330.23261470.22188919X-RAY DIFFRACTION99
3.9633-4.26920.25131450.19988871X-RAY DIFFRACTION98
4.2692-4.69850.19181430.17198777X-RAY DIFFRACTION96
4.6985-5.37770.25081460.18249010X-RAY DIFFRACTION99
5.3777-6.77250.22351450.21158839X-RAY DIFFRACTION96
6.7725-49.44570.19011470.18329039X-RAY DIFFRACTION97

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