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

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Basic information

Entry
Database: PDB / ID: 6ojs
TitleCrystal structure of TS-DHFR from Cryptosporidium hominis in complex with NADPH, FdUMP, MTX and 2-amino-4-oxo-4,7-dihydro-pyrrolo[2,3-d]pyrimidine-methyl-phenyl-D-glutamic 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
Chem-D96 / METHOTREXATE / Chem-NDP / 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: 3.214 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: Febs Lett. / Year: 2019
Title: Understanding the structural basis of species selective, stereospecific inhibition for Cryptosporidium and human thymidylate synthase.
Authors: Czyzyk, D.J. / Valhondo, M. / Jorgensen, W.L. / Anderson, K.S.
History
DepositionApr 12, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 19, 2019Provider: repository / Type: Initial release
Revision 1.1Aug 21, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Oct 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)311,01025
Polymers301,3135
Non-polymers9,69720
Water00
1
A: Bifunctional dihydrofolate reductase-thymidylate synthase
C: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,40410
Polymers120,5252
Non-polymers3,8798
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14180 Å2
ΔGint-48 kcal/mol
Surface area37630 Å2
MethodPISA
2
B: Bifunctional dihydrofolate reductase-thymidylate synthase
E: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,40410
Polymers120,5252
Non-polymers3,8798
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area14230 Å2
ΔGint-51 kcal/mol
Surface area37690 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)124,40410
Polymers120,5252
Non-polymers3,8798
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y,-z1
Buried area14400 Å2
ΔGint-45 kcal/mol
Surface area36680 Å2
MethodPISA
Unit cell
Length a, b, c (Å)212.622, 116.188, 220.884
Angle α, β, γ (deg.)90.00, 95.48, 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-D96 / N-{4-[(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl]benzene-1-carbonyl}-D-glutamic acid


Mass: 413.384 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C19H19N5O6
#5: Chemical
ChemComp-MTX / METHOTREXATE


Mass: 454.439 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C20H22N8O5

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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 22% 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: 0.97915 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jul 6, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97915 Å / Relative weight: 1
ReflectionResolution: 3.21→51 Å / Num. obs: 85851 / % possible obs: 98.2 % / Redundancy: 6.9 % / CC1/2: 0.983 / Rsym value: 0.317 / Net I/σ(I): 4.98
Reflection shellResolution: 3.21→3.41 Å / Redundancy: 6.7 % / Mean I/σ(I) obs: 1.06 / Num. unique obs: 12774 / CC1/2: 0.616 / Rsym value: 1.734 / % possible all: 91.1

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Processing

Software
NameVersionClassification
PHENIX(1.10.1_2155)refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4Q0D

4q0d


Resolution: 3.214→50.121 Å / SU ML: 0.45 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 27.19
RfactorNum. reflection% reflection
Rfree0.2317 4282 5 %
Rwork0.2179 --
obs0.2186 85687 98.03 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 3.214→50.121 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms20266 0 660 0 20926
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00321446
X-RAY DIFFRACTIONf_angle_d0.50229177
X-RAY DIFFRACTIONf_dihedral_angle_d10.0812445
X-RAY DIFFRACTIONf_chiral_restr0.0433135
X-RAY DIFFRACTIONf_plane_restr0.0033731
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.2142-3.25070.4376830.43131576X-RAY DIFFRACTION57
3.2507-3.2890.36411450.36022748X-RAY DIFFRACTION99
3.289-3.32910.36311440.35882731X-RAY DIFFRACTION100
3.3291-3.37120.34641450.34312751X-RAY DIFFRACTION99
3.3712-3.41550.33431450.33372748X-RAY DIFFRACTION100
3.4155-3.46230.41031440.35022751X-RAY DIFFRACTION100
3.4623-3.51180.32191430.30842722X-RAY DIFFRACTION100
3.5118-3.56420.32721460.29812788X-RAY DIFFRACTION100
3.5642-3.61990.34281410.29432695X-RAY DIFFRACTION100
3.6199-3.67920.29791460.27762763X-RAY DIFFRACTION100
3.6792-3.74260.28451430.26272717X-RAY DIFFRACTION99
3.7426-3.81060.22991430.24912720X-RAY DIFFRACTION100
3.8106-3.88390.25271460.25332768X-RAY DIFFRACTION100
3.8839-3.96320.2741440.25042744X-RAY DIFFRACTION99
3.9632-4.04930.23741460.23472756X-RAY DIFFRACTION100
4.0493-4.14340.26431430.22912729X-RAY DIFFRACTION100
4.1434-4.2470.21881450.20142757X-RAY DIFFRACTION99
4.247-4.36180.20911440.19712744X-RAY DIFFRACTION100
4.3618-4.490.20711460.19112766X-RAY DIFFRACTION99
4.49-4.63490.2051410.18312676X-RAY DIFFRACTION97
4.6349-4.80040.18551440.18042730X-RAY DIFFRACTION100
4.8004-4.99240.20081460.17552782X-RAY DIFFRACTION100
4.9924-5.21940.20681460.18752766X-RAY DIFFRACTION100
5.2194-5.49430.20621460.1962782X-RAY DIFFRACTION100
5.4943-5.8380.23571460.2092767X-RAY DIFFRACTION100
5.838-6.2880.21811450.2082761X-RAY DIFFRACTION100
6.288-6.91930.22491460.20262777X-RAY DIFFRACTION100
6.9193-7.91720.19721460.17932790X-RAY DIFFRACTION100
7.9172-9.9620.14371450.15052740X-RAY DIFFRACTION97
9.962-50.12720.19141490.1822860X-RAY DIFFRACTION99

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