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- PDB-1kms: HUMAN DIHYDROFOLATE REDUCTASE COMPLEXED WITH NADPH AND 6-([5-QUIN... -

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Basic information

Entry
Database: PDB / ID: 1kms
TitleHUMAN DIHYDROFOLATE REDUCTASE COMPLEXED WITH NADPH AND 6-([5-QUINOLYLAMINO]METHYL)-2,4-DIAMINO-5-METHYLPYRIDO[2,3-D]PYRIMIDINE (SRI-9439), A LIPOPHILIC ANTIFOLATE
ComponentsDIHYDROFOLATE REDUCTASE
KeywordsOXIDOREDUCTASE / ANTIPARASITIC DRUGS / REDUCTASE / LIPOPHILIC ANTIFOLATES
Function / homology
Function and homology information


regulation of removal of superoxide radicals / tetrahydrobiopterin biosynthetic process / Metabolism of folate and pterines / tetrahydrofolate metabolic process / response to methotrexate / sequence-specific mRNA binding / folic acid binding / axon regeneration / dihydrofolate metabolic process / G1/S-Specific Transcription ...regulation of removal of superoxide radicals / tetrahydrobiopterin biosynthetic process / Metabolism of folate and pterines / tetrahydrofolate metabolic process / response to methotrexate / sequence-specific mRNA binding / folic acid binding / axon regeneration / dihydrofolate metabolic process / G1/S-Specific Transcription / dihydrofolate reductase / dihydrofolate reductase activity / folic acid metabolic process / NADPH binding / tetrahydrofolate biosynthetic process / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / one-carbon metabolic process / mRNA regulatory element binding translation repressor activity / NADP binding / negative regulation of translation / mRNA binding / mitochondrion / cytosol
Similarity search - Function
Dihydrofolate reductase / 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 / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Chem-LIH / Chem-NDP / Dihydrofolate reductase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.09 Å
AuthorsKlon, A.E. / Heroux, A. / Ross, L.J. / Pathak, V. / Johnson, C.A. / Piper, J.R. / Borhani, D.W.
CitationJournal: J.Mol.Biol. / Year: 2002
Title: Atomic structures of human dihydrofolate reductase complexed with NADPH and two lipophilic antifolates at 1.09 a and 1.05 a resolution.
Authors: Klon, A.E. / Heroux, A. / Ross, L.J. / Pathak, V. / Johnson, C.A. / Piper, J.R. / Borhani, D.W.
History
DepositionDec 17, 2001Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 10, 2002Provider: repository / Type: Initial release
Revision 1.1Apr 27, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Feb 14, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.4Apr 3, 2024Group: Refinement description / Category: pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DIHYDROFOLATE REDUCTASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)22,6185
Polymers21,3501
Non-polymers1,2694
Water7,188399
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)41.467, 55.275, 82.799
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein DIHYDROFOLATE REDUCTASE / DHFR


Mass: 21349.525 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: PDFR / Species (production host): Escherichia coli / Cellular location (production host): CYTOPLASM / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P00374, dihydrofolate reductase
#2: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4
#3: Chemical ChemComp-LIH / 6-([5-QUINOLYLAMINO]METHYL)-2,4-DIAMINO-5-METHYLPYRIDO[2,3-D]PYRIMIDINE / SRI-9439


Mass: 331.374 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C18H17N7
#4: Chemical ChemComp-NDP / NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE


Mass: 745.421 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H30N7O17P3
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 399 / 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: 2.23 Å3/Da / Density % sol: 46 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 8
Details: THE TERNARY COMPLEX OF DHFR WITH NADPH AND SRI-9439 WAS FORMED BY MIXING HUMAN DHFR (20 MG/ML IN 25 MM KPO4 (PH 7.0), 0.1 MM EDTA, AND 3 MM NAN3) WITH 60 MM NADPH, FOLLOWED 15 MINUTES LATER ...Details: THE TERNARY COMPLEX OF DHFR WITH NADPH AND SRI-9439 WAS FORMED BY MIXING HUMAN DHFR (20 MG/ML IN 25 MM KPO4 (PH 7.0), 0.1 MM EDTA, AND 3 MM NAN3) WITH 60 MM NADPH, FOLLOWED 15 MINUTES LATER BY 60 MM OF INHIBITOR IN DMSO (FINAL CONCENTRATIONS OF 2 MM NADPH AND 2 MM INHIBITOR). THIS COMPLEX SOLUTION WAS MIXED WITH AN EQUAL VOLUME OF PRECIPITANT CONTAINING 24-33% PEG 4000, 0.2 M LI2SO4, 0.1 M TRIS CL (PH 7.9-8.4), AND EQUILIBRATED WITH THE PRECIPITANT BY HANGING DROP VAPOR DIFFUSION AT 277 K. THE CRYSTAL GREW IN ABOUT 3 WEEKS. THE CRYSTAL WAS FLASH-COOLED DIRECTLY IN LIQUID NITROGEN AFTER HARVESTING INTO MOTHER LIQUOR CONTAINING 10% GLYCEROL., pH 8.00, VAPOR DIFFUSION, HANGING DROP
Crystal grow
*PLUS
Temperature: 4 ℃ / pH: 7
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDDetailsChemical formula
120 mg/mlhDHFR1drop
225 mM1droppH7.0KPO4
30.1 mMEDTA1drop
43 mM1dropNaN3
52 mMNADPH1drop
62 mMinhibitor1drop
724-33 %(w/v)PEG40001reservoir
80.2 M1reservoirLi2SO4
90.1 MTris-HCl1reservoirpH7.9-8.4

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL7-1 / Wavelength: 1.08
DetectorType: MARRESEARCH / Detector: IMAGE PLATE / Date: Jun 25, 1997 / Details: PT-COATED MIRROR
RadiationMonochromator: SI (111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.08 Å / Relative weight: 1
ReflectionResolution: 1.09→20 Å / Num. all: 79611 / Num. obs: 79611 / % possible obs: 99.6 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 3.6 % / Biso Wilson estimate: 10.8 Å2 / Rmerge(I) obs: 0.047 / Rsym value: 0.047 / Net I/σ(I): 9.4
Reflection shellResolution: 1.09→1.15 Å / Redundancy: 3.3 % / Rmerge(I) obs: 0.422 / Mean I/σ(I) obs: 2.3 / Rsym value: 0.422 / % possible all: 99.1
Reflection
*PLUS
Highest resolution: 1.09 Å / Lowest resolution: 20 Å / Num. measured all: 289350 / Rmerge(I) obs: 0.047
Reflection shell
*PLUS
% possible obs: 99.1 % / Rmerge(I) obs: 0.422

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Processing

Software
NameVersionClassification
AMoREphasing
SHELXL-97refinement
MOSFLMdata reduction
CCP4(SCALA)data scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: UNPUBLISHED HUMAN DHFR STRUCTURE.

Resolution: 1.09→20 Å / Num. parameters: 18984 / Num. restraintsaints: 23468 / Cross valid method: THROUGHOUT / σ(F): 0 / σ(I): 0 / Stereochemistry target values: ENGH & HUBER
Details: REFINEMENT IN X-PLOR, ALTERNATED WITH MANUAL REBUILDING IN O, RESULTING IN A FREE R-FACTOR OF 24.0%. SRI-9439 WAS THEN ADDED TO THE MODEL IN THE ACTIVE SITE AND THE CIS PEPTIDE BONDS BETWEEN ...Details: REFINEMENT IN X-PLOR, ALTERNATED WITH MANUAL REBUILDING IN O, RESULTING IN A FREE R-FACTOR OF 24.0%. SRI-9439 WAS THEN ADDED TO THE MODEL IN THE ACTIVE SITE AND THE CIS PEPTIDE BONDS BETWEEN RESIDUES ARG-65 AND PRO-66 AND RESIDUES GLY-116 AND GLY-117 BECAME APPARENT IN 2FO-FC AND FO-FC MAPS. REFINEMENT PROCEEDED WITH THE ADDITION OF RIDING HYDROGEN ATOMS AND ANISOTROPIC TEMPERATURE FACTORS IN REFMAC AND ARP, YIELDING A FREE R-FACTOR OF 19.4%. AT THIS POINT, MOST OF THE SIDE CHAIN ALTERNATE CONFORMATIONS WERE MODELED. ANISOTROPIC DISPLACEMENT PARAMETERS WERE REFINED IN SHELXL AND RIDING HYDROGEN ATOMS WERE ADDED. IN THE FINAL ROUNDS OF REFINEMENT, RESTRAINTS FOR ALL ATOMS IN SRI-9439 EXCEPT FOR PLANARITY RESTRAINTS ON THE 5-QUINOLYLAMINO GROUP, AND ALL NADPH ATOMS EXCEPT FOR THOSE IN THE ADENINE RING AND ADENINE RIBOSE-2-PHOSPHATE WERE REMOVED, GIVING THE FINAL FREE R-FACTOR (17.3%). ATTEMPTS TO RESTRAIN THE PLANARITY OF THE 5-METHYL-5-DEAZAPTERIDINE MOIETY OF SRI-9439 RESULTED IN LARGE DIFFERENCE ELECTRON DENSITY PEAKS FOR THE EXOCYCLIC N4 AND C5A ATOMS, CONFIRMING THEIR OUT-OF-PLANE POSITIONS.
RfactorNum. reflection% reflectionSelection details
Rfree0.1726 3988 5 %5% OF THE REFLECTIONS WERE RANDOMLY SELECTED FOR THE TEST SET.
Rwork0.1306 ---
all-79411 --
obs-79411 99.6 %-
Solvent computationSolvent model: MOEWS & KRETSINGER, J.MOL.BIOL.91(1973)201-228
Refine analyzeNum. disordered residues: 66 / Occupancy sum hydrogen: 1406.12 / Occupancy sum non hydrogen: 1927.22
Refinement stepCycle: LAST / Resolution: 1.09→20 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1606 0 83 399 2088
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONs_bond_d0.013
X-RAY DIFFRACTIONs_angle_d0.032
X-RAY DIFFRACTIONs_similar_dist0
X-RAY DIFFRACTIONs_from_restr_planes0.0293
X-RAY DIFFRACTIONs_zero_chiral_vol0.076
X-RAY DIFFRACTIONs_non_zero_chiral_vol0.086
X-RAY DIFFRACTIONs_anti_bump_dis_restr0.023
X-RAY DIFFRACTIONs_rigid_bond_adp_cmpnt0.005
X-RAY DIFFRACTIONs_similar_adp_cmpnt0.05
X-RAY DIFFRACTIONs_approx_iso_adps0.098
Software
*PLUS
Name: SHELXL / Version: 97 / Classification: refinement
Refinement
*PLUS
% reflection Rfree: 5 % / Rfactor Rfree: 0.173 / Rfactor Rwork: 0.131
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONs_angle_d
X-RAY DIFFRACTIONs_angle_deg2.23

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