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Yorodumi- PDB-1kms: HUMAN DIHYDROFOLATE REDUCTASE COMPLEXED WITH NADPH AND 6-([5-QUIN... -
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-Basic information
Entry | Database: PDB / ID: 1kms | ||||||
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Title | HUMAN DIHYDROFOLATE REDUCTASE COMPLEXED WITH NADPH AND 6-([5-QUINOLYLAMINO]METHYL)-2,4-DIAMINO-5-METHYLPYRIDO[2,3-D]PYRIMIDINE (SRI-9439), A LIPOPHILIC ANTIFOLATE | ||||||
Components | DIHYDROFOLATE REDUCTASE | ||||||
Keywords | OXIDOREDUCTASE / 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 / axon regeneration / folic acid binding / G1/S-Specific Transcription / dihydrofolate metabolic process ...regulation of removal of superoxide radicals / tetrahydrobiopterin biosynthetic process / Metabolism of folate and pterines / tetrahydrofolate metabolic process / response to methotrexate / sequence-specific mRNA binding / axon regeneration / folic acid binding / G1/S-Specific Transcription / dihydrofolate metabolic process / dihydrofolate reductase / dihydrofolate reductase activity / folic acid metabolic process / NADPH binding / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / tetrahydrofolate biosynthetic process / mRNA regulatory element binding translation repressor activity / positive regulation of nitric-oxide synthase activity / one-carbon metabolic process / NADP binding / negative regulation of translation / mRNA binding / mitochondrion / cytosol Similarity search - Function | ||||||
Biological species | Homo sapiens (human) | ||||||
Method | X-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.09 Å | ||||||
Authors | Klon, A.E. / Heroux, A. / Ross, L.J. / Pathak, V. / Johnson, C.A. / Piper, J.R. / Borhani, D.W. | ||||||
Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 1kms.cif.gz | 116.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb1kms.ent.gz | 90.3 KB | Display | PDB format |
PDBx/mmJSON format | 1kms.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/km/1kms ftp://data.pdbj.org/pub/pdb/validation_reports/km/1kms | HTTPS FTP |
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-Related structure data
-Links
-Assembly
Deposited unit |
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1 |
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Unit cell |
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-Components
#1: Protein | 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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#2: Chemical | #3: Chemical | ChemComp-LIH / | #4: Chemical | ChemComp-NDP / | #5: Water | ChemComp-HOH / | |
-Experimental details
-Experiment
Experiment | Method: X-RAY DIFFRACTION / Number of used crystals: 1 |
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-Sample preparation
Crystal | Density Matthews: 2.23 Å3/Da / Density % sol: 46 % | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Crystal grow | Temperature: 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
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-Data collection
Diffraction | Mean temperature: 100 K |
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Diffraction source | Source: SYNCHROTRON / Site: SSRL / Beamline: BL7-1 / Wavelength: 1.08 |
Detector | Type: MARRESEARCH / Detector: IMAGE PLATE / Date: Jun 25, 1997 / Details: PT-COATED MIRROR |
Radiation | Monochromator: SI (111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray |
Radiation wavelength | Wavelength: 1.08 Å / Relative weight: 1 |
Reflection | Resolution: 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 shell | Resolution: 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 |
-Processing
Software |
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Refinement | Method 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.
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Solvent computation | Solvent model: MOEWS & KRETSINGER, J.MOL.BIOL.91(1973)201-228 | |||||||||||||||||||||||||||||||||
Refine analyze | Num. disordered residues: 66 / Occupancy sum hydrogen: 1406.12 / Occupancy sum non hydrogen: 1927.22 | |||||||||||||||||||||||||||||||||
Refinement step | Cycle: LAST / Resolution: 1.09→20 Å
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Refine LS restraints |
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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
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