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- PDB-1kmv: HUMAN DIHYDROFOLATE REDUCTASE COMPLEXED WITH NADPH AND (Z)-6-(2-[... -

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Basic information

Entry
Database: PDB / ID: 1kmv
TitleHUMAN DIHYDROFOLATE REDUCTASE COMPLEXED WITH NADPH AND (Z)-6-(2-[2,5-DIMETHOXYPHENYL]ETHEN-1-YL)-2,4-DIAMINO-5-METHYLPYRIDO[2,3-D]PYRIMIDINE (SRI-9662), 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 / tetrahydrofolate metabolic process / methotrexate binding / response to methotrexate / dihydrofolate metabolic process / sequence-specific mRNA binding / axon regeneration / folic acid binding / folic acid metabolic process ...regulation of removal of superoxide radicals / tetrahydrobiopterin biosynthetic process / tetrahydrofolate metabolic process / methotrexate binding / response to methotrexate / dihydrofolate metabolic process / sequence-specific mRNA binding / axon regeneration / folic acid binding / folic acid metabolic process / glycine biosynthetic process / dihydrofolate reductase / dihydrofolate reductase activity / regulation of transcription involved in G1/S transition of mitotic cell cycle / translation repressor activity, mRNA regulatory element binding / NADPH binding / tetrahydrofolate biosynthetic process / drug binding / positive regulation of nitric-oxide synthase activity / one-carbon metabolic process / NADP binding / negative regulation of translation / mRNA binding / mitochondrion / cytosol
Dihydrofolate reductase-like domain superfamily / Dihydrofolate reductase / Dihydrofolate reductase conserved site / Dihydrofolate reductase / Dihydrofolate reductase domain / Dihydrofolate Reductase, subunit A / Dihydrofolate Reductase, subunit A / 3-Layer(aba) Sandwich / Alpha Beta
Dihydrofolate reductase
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.05 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.
Validation Report
SummaryFull reportAbout validation report
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

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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)22,6075
Polymers21,3501
Non-polymers1,2574
Water6,089338
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
γ
α
β
Length a, b, c (Å)41.300, 54.990, 82.680
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

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Protein , 1 types, 1 molecules A

#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

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Non-polymers , 5 types, 342 molecules

#2: Chemical ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#3: Chemical ChemComp-LII / (Z)-6-(2-[2,5-DIMETHOXYPHENYL]ETHEN-1-YL)-2,4-DIAMINO-5-METHYLPYRIDO[2,3-D]PYRIMIDINE / SRI-9662


Mass: 337.376 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C18H19N5O2
#4: Chemical ChemComp-NDP / NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Nicotinamide adenine dinucleotide phosphate


Mass: 745.421 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H30N7O17P3
#5: Chemical ChemComp-DMS / DIMETHYL SULFOXIDE / Dimethyl sulfoxide


Mass: 78.133 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6OS / Comment: DMSO, precipitant*YM
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 338 / 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: 2.2 Å3/Da / Density % sol: 45 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 8
Details: THE TERNARY COMPLEX OF DHFR WITH NADPH AND SRI-9662 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-9662 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), 5% GLYCEROL, 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
105 %(v/v)glycerol1reservoirfor hDHFR/NADPH/SRI-9662 ternary complex

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X25 / Wavelength: 0.9
DetectorType: BRANDEIS - B4 / Detector: CCD / Date: Jun 18, 1998 / Details: PT-COATED MIRROR
RadiationMonochromator: SI (111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9 Å / Relative weight: 1
ReflectionResolution: 1.05→20 Å / Num. obs: 86349 / % possible obs: 97.5 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 3.6 % / Biso Wilson estimate: 8.2 Å2 / Rmerge(I) obs: 0.053 / Rsym value: 0.053 / Net I/σ(I): 9.6
Reflection shellResolution: 1.05→1.09 Å / Redundancy: 2.1 % / Rmerge(I) obs: 0.231 / Mean I/σ(I) obs: 2.8 / Rsym value: 0.231 / % possible all: 83.8
Reflection
*PLUS
Highest resolution: 1.05 Å / Lowest resolution: 20 Å / Num. measured all: 312507 / Rmerge(I) obs: 0.053
Reflection shell
*PLUS
% possible obs: 83.8 % / Rmerge(I) obs: 0.231

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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: 1.05→20 Å / Num. parameters: 17886 / Num. restraintsaints: 21912 / Cross valid method: THROUGHOUT / σ(F): 0 / σ(I): 0 / Stereochemistry target values: ENGH & HUBER
Details: REFINEMENT BEGAN WITH RIGID-BODY MINIMIZATION (X-PLOR) USING THE COORDINATES AND ISOTROPIC TEMPERATURE FACTORS OF HDHFR AND NADPH FROM THE HDHFR/NADPH/SRI-9439 TERNARY COMPLEX (PDB ENTRY ...Details: REFINEMENT BEGAN WITH RIGID-BODY MINIMIZATION (X-PLOR) USING THE COORDINATES AND ISOTROPIC TEMPERATURE FACTORS OF HDHFR AND NADPH FROM THE HDHFR/NADPH/SRI-9439 TERNARY COMPLEX (PDB ENTRY 1KMS), WITH THE CIS PEPTIDE BONDS RESTRAINED. ALTERNATING BETWEEN REFINEMENT IN X-PLOR AND MANUAL REBUILDING IN O RESULTED IN A FREE R-FACTOR OF 23.9%. SRI-9662 WAS ADDED AFTER ITS DENSITY BECAME APPARENT IN THE 2FO-FC AND FO-FC MAPS. REFINEMENT CONTINUED WITH THE ADDITION OF RIDING HYDROGEN ATOMS AND ANISOTROPIC TEMPERATURE FACTORS IN REFMAC AND ARP, RESULTING IN A FREE R-FACTOR OF 19.6%. MOST OF THE SIDE CHAINS WITH ALTERNATE CONFORMATIONS WERE MODELED AT THIS POINT. REFINEMENT WAS COMPLETED USING SHELXL. REFINEMENT OF THE ANISOTROPIC DISPLACEMENT PARAMETERS, ADDITION OF RIDING HYDROGENS, AND REMOVAL OF ALL RESTRAINTS ON THE PTERIDINE RING OF SRI-9662 RESULTED IN A FREE R-FACTOR OF 18.1%. SEVERAL LARGE PEAKS OF POSITIVE DIFFERENCE DENSITY WERE LOCATED IN THE NADPH BINDING SITE, THE LARGEST BEING 0.7 A AWAY FROM THE C7N CARBON ATOM, AND WERE ASSIGNED TO DMSO AT PARTIAL OCCUPANCY. HOH 607, 608, AND 609 ARE LOCATED IN THE NDP BINDING SITE, ARE PRESENT ONLY WHEN NDP IS NOT, AND ARE THUS INCLUDED IN ALTERNATE CONFORMATION B, ALONG WITH THE DMSO MOLECULE (NDP IS MODELED AS ALTERNATE CONFORMATION A).
RfactorNum. reflection% reflectionSelection details
Rfree0.1807 4325 5 %5% OF THE REFLECTIONS WERE RANDOMLY SELECTED FOR THE TEST SET.
Rwork0.1302 ---
All-86271 --
Obs-86271 97.5 %-
Solvent computationSolvent model: MOEWS & KRETSINGER, J.MOL.BIOL.91(1973)201-228
Refine analyzeNum. disordered residues: 17 / Occupancy sum hydrogen: 1412.08 / Occupancy sum non hydrogen: 1843.16
Refinement stepCycle: LAST / Resolution: 1.05→20 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1528 0 82 338 1948
Refine LS restraints
Refinement-IDTypeDev ideal
X-RAY DIFFRACTIONs_bond_d0.017
X-RAY DIFFRACTIONs_angle_d0.035
X-RAY DIFFRACTIONs_similar_dist0
X-RAY DIFFRACTIONs_from_restr_planes0.029
X-RAY DIFFRACTIONs_zero_chiral_vol0.081
X-RAY DIFFRACTIONs_non_zero_chiral_vol0.086
X-RAY DIFFRACTIONs_anti_bump_dis_restr0.031
X-RAY DIFFRACTIONs_rigid_bond_adp_cmpnt0.006
X-RAY DIFFRACTIONs_similar_adp_cmpnt0.058
X-RAY DIFFRACTIONs_approx_iso_adps0.105
Software
*PLUS
Name: SHELXL / Version: 97 / Classification: refinement
Refinement
*PLUS
% reflection Rfree: 5 % / Rfactor Rfree: 0.181 / Rfactor Rwork: 0.13
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Type: s_angle_d / Dev ideal: 2.27

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