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- PDB-4q0e: Crystal structure of TS-DHFR from Cryptosporidium hominis in comp... -

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Basic information

Entry
Database: PDB / ID: 4q0e
TitleCrystal structure of TS-DHFR from Cryptosporidium hominis in complex with NADPH, FdUMP and 2-amino-4-oxo-4,7-dihydro-pyrrolo[2,3-d]pyrimidine-methyl-phenyl-L-glutamic acid.
ComponentsBifunctional dihydrofolate reductase-thymidylate synthase
Keywordstransferase/transferase inhibitor / bifunctional enzyme / transferase / oxidoreductase / transferase-transferase inhibitor complex
Function / homology
Function and homology information


Thymidylate Synthase; Chain A / Thymidylate synthase/dCMP hydroxymethylase domain / Dihydrofolate Reductase, subunit A / Dihydrofolate Reductase, subunit A / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Chem-2XB / Chem-NDP / 5-FLUORO-2'-DEOXYURIDINE-5'-MONOPHOSPHATE / :
Similarity search - Component
Biological speciesCryptosporidium hominis (eukaryote)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.78 Å
AuthorsKumar, V.P. / Anderson, K.S.
CitationJournal: Bioorg.Med.Chem.Lett. / Year: 2014
Title: Structural studies provide clues for analog design of specific inhibitors of Cryptosporidium hominis thymidylate synthase-dihydrofolate reductase.
Authors: Kumar, V.P. / Cisneros, J.A. / Frey, K.M. / Castellanos-Gonzalez, A. / Wang, Y. / Gangjee, A. / White, A.C. / Jorgensen, W.L. / Anderson, K.S.
History
DepositionApr 1, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 15, 2014Provider: repository / Type: Initial release
Revision 1.1Nov 22, 2017Group: Refinement description / Category: software
Revision 1.2Sep 20, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_ncs_dom_lim / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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)310,80425
Polymers301,3135
Non-polymers9,49220
Water32418
1
A: Bifunctional dihydrofolate reductase-thymidylate synthase
C: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,32210
Polymers120,5252
Non-polymers3,7978
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11910 Å2
ΔGint-38 kcal/mol
Surface area38150 Å2
MethodPISA
2
B: Bifunctional dihydrofolate reductase-thymidylate synthase
D: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)124,32210
Polymers120,5252
Non-polymers3,7978
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11830 Å2
ΔGint-40 kcal/mol
Surface area38330 Å2
MethodPISA
3
E: Bifunctional dihydrofolate reductase-thymidylate synthase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)62,1615
Polymers60,2631
Non-polymers1,8984
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)213.629, 115.540, 217.973
Angle α, β, γ (deg.)90.00, 94.56, 90.00
Int Tables number5
Space group name H-MC121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain A and (resseq 3:178 or resseq 193:521 )
21chain B and (resseq 3:178 or resseq 193:521 )
31chain C and (resseq 3:178 or resseq 193:521 )
41chain D and (resseq 3:178 or resseq 193:521 )
51chain E and (resseq 3:178 or resseq 193:521 )

NCS domain segments:

Ens-ID: 1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11GLUGLUGLNGLNchain A and (resseq 3:178 or resseq 193:521 )AA3 - 1783 - 178
12LEULEUVALVALchain A and (resseq 3:178 or resseq 193:521 )AA193 - 521193 - 521
21GLUGLUGLNGLNchain B and (resseq 3:178 or resseq 193:521 )BB3 - 1783 - 178
22LEULEUVALVALchain B and (resseq 3:178 or resseq 193:521 )BB193 - 521193 - 521
31GLUGLUGLNGLNchain C and (resseq 3:178 or resseq 193:521 )CC3 - 1783 - 178
32LEULEUVALVALchain C and (resseq 3:178 or resseq 193:521 )CC193 - 521193 - 521
41GLUGLUGLNGLNchain D and (resseq 3:178 or resseq 193:521 )DD3 - 1783 - 178
42LEULEUVALVALchain D and (resseq 3:178 or resseq 193:521 )DD193 - 521193 - 521
51GLUGLUGLNGLNchain E and (resseq 3:178 or resseq 193:521 )EE3 - 1783 - 178
52LEULEUVALVALchain E and (resseq 3:178 or resseq 193:521 )EE193 - 521193 - 521

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Components

#1: Protein
Bifunctional dihydrofolate reductase-thymidylate synthase


Mass: 60262.520 Da / Num. of mol.: 5
Fragment: bifunctional thymidylate synthase-dihydrofolate reductase
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cryptosporidium hominis (eukaryote) / Gene: Chro.40506, TS-DHFR / Plasmid: pTrc99A / Production host: Escherichia coli (E. coli) / Strain (production host): PA414
References: UniProt: Q5CGA3, thymidylate synthase, dihydrofolate reductase
#2: Chemical
ChemComp-NDP / NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / 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 / Fluorodeoxyuridylate


Type: DNA linking / Mass: 326.172 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C9H12FN2O8P / Comment: inhibitor*YM
#4: Chemical
ChemComp-2XB / N-{4-[(2-amino-4-hydroxy-7H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl]benzoyl}-L-glutamic acid


Mass: 413.384 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C19H19N5O6
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 18 / Source method: isolated from a natural source / Formula: H2O

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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: 4.45 Å3/Da / Density % sol: 72.36 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 12% (w/v) PEG 6000, 60 mM ammonium sulfate, 200 mM lithium sulfate, and 100 mM Tris pH 8.0. ChTS-DHFR enzyme (approximately 7 mg/ml) with 1 mM NADPH, 1 mM FdUMP and 0.5 mM inhibitor., VAPOR ...Details: 12% (w/v) PEG 6000, 60 mM ammonium sulfate, 200 mM lithium sulfate, and 100 mM Tris pH 8.0. ChTS-DHFR enzyme (approximately 7 mg/ml) with 1 mM NADPH, 1 mM FdUMP and 0.5 mM inhibitor., VAPOR DIFFUSION, HANGING DROP, temperature 298K

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Data collection

DiffractionMean temperature: 77 K
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X29A / Wavelength: 1.075 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Oct 18, 2013 / Details: Monochromator
RadiationMonochromator: Cryogenically cooled crystal monochromator with horizontal focusing sagittal bend second mono crystal with 4:1 magnification ratio and vertically focusing mirror.
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.075 Å / Relative weight: 1
ReflectionResolution: 2.76→50 Å / Num. all: 427317 / Num. obs: 124124 / % possible obs: 94.1 % / Observed criterion σ(I): -3 / Redundancy: 3.5 % / Biso Wilson estimate: 56.74 Å2 / Rmerge(I) obs: 0.132 / Rsym value: 0.132 / Net I/σ(I): 12.8
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsDiffraction-ID% possible all
2.76-2.813.40.522194.2
2.81-2.863.50.463194.1
2.86-2.913.50.459194.6
2.91-2.973.50.351194.6
2.97-3.043.50.312194.1
3.04-3.113.50.287193.5
3.11-3.193.50.251193.9
3.19-3.273.50.228193.3
3.27-3.373.50.2193.4
3.37-3.483.50.2193.1
3.48-3.63.50.166193.3
3.6-3.753.50.16192.8
3.75-3.923.50.156192.5
3.92-4.123.50.132192.8
4.12-4.383.50.123192.1
4.38-4.723.50.12192.6
4.72-5.193.50.112192.8
5.19-5.943.50.104195.5
5.94-7.483.50.089199.4
7.48-503.70.068198.7

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
SCALEPACKdata scaling
PHENIX1.8.4_1496refinement
PDB_EXTRACT3.14data extraction
CBASSdata collection
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: pdb entry 4KY8

4ky8


Resolution: 2.78→48.054 Å / SU ML: 0.39 / σ(F): 1.34 / Phase error: 31.15 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2703 2015 1.62 %
Rwork0.2549 --
obs0.2551 124074 93.22 %
all-124124 -
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 64.11 Å2
Refinement stepCycle: LAST / Resolution: 2.78→48.054 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms20491 0 645 18 21154
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00421656
X-RAY DIFFRACTIONf_angle_d0.7329375
X-RAY DIFFRACTIONf_dihedral_angle_d14.588162
X-RAY DIFFRACTIONf_chiral_restr0.0443110
X-RAY DIFFRACTIONf_plane_restr0.0043739
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDTypeRms dev position (Å)
11A4095X-RAY DIFFRACTIONPOSITIONAL0.156
12B4095X-RAY DIFFRACTIONPOSITIONAL0.156
13C4099X-RAY DIFFRACTIONPOSITIONAL0.182
14D4099X-RAY DIFFRACTIONPOSITIONAL0.158
15E4099X-RAY DIFFRACTIONPOSITIONAL0.193
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.7796-2.84910.36411470.35447651X-RAY DIFFRACTION83
2.8491-2.92610.38111370.36968787X-RAY DIFFRACTION94
2.9261-3.01220.42471450.36778780X-RAY DIFFRACTION94
3.0122-3.10940.38971400.36028780X-RAY DIFFRACTION94
3.1094-3.22050.33071310.34148724X-RAY DIFFRACTION94
3.2205-3.34950.32421460.32618703X-RAY DIFFRACTION94
3.3495-3.50190.31671570.30328674X-RAY DIFFRACTION93
3.5019-3.68640.28081420.27888707X-RAY DIFFRACTION93
3.6864-3.91730.26751390.25868671X-RAY DIFFRACTION93
3.9173-4.21960.27061400.23238605X-RAY DIFFRACTION93
4.2196-4.64390.21851420.19458660X-RAY DIFFRACTION92
4.6439-5.31510.20111410.19638723X-RAY DIFFRACTION93
5.3151-6.69360.24581500.22569126X-RAY DIFFRACTION97
6.6936-48.06130.2171580.20749468X-RAY DIFFRACTION99

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