1KMS
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
Experimental procedure
| Experimental method | SINGLE WAVELENGTH |
| Source type | SYNCHROTRON |
| Source details | SSRL BEAMLINE BL7-1 |
| Synchrotron site | SSRL |
| Beamline | BL7-1 |
| Temperature [K] | 100 |
| Detector technology | IMAGE PLATE |
| Collection date | 1997-06-25 |
| Detector | MARRESEARCH |
| Spacegroup name | P 21 21 21 |
| Unit cell lengths | 41.467, 55.275, 82.799 |
| Unit cell angles | 90.00, 90.00, 90.00 |
Refinement procedure
| Resolution | 20.000 - 1.090 |
| Rwork | 0.131 |
| R-free | 0.17300 * |
| Structure solution method | MOLECULAR REPLACEMENT |
| Starting model (for MR) | UNPUBLISHED HUMAN DHFR STRUCTURE. |
| RMSD bond length | 0.013 |
| RMSD bond angle | 2.230 * |
| Data reduction software | MOSFLM |
| Data scaling software | CCP4 ((SCALA)) |
| Phasing software | AMoRE |
| Refinement software | SHELXL-97 |
Data quality characteristics
| Overall | Outer shell | |
| Low resolution limit [Å] | 20.000 | 1.150 |
| High resolution limit [Å] | 1.090 | 1.090 |
| Rmerge | 0.047 | 0.422 |
| Total number of observations | 289350 * | |
| Number of reflections | 79611 | |
| <I/σ(I)> | 9.4 | 2.3 |
| Completeness [%] | 99.6 | 99.1 |
| Redundancy | 3.6 | 3.3 |
Crystallization Conditions
| crystal ID | method | pH | temperature | details |
| 1 | VAPOR DIFFUSION, HANGING DROP | 7 * | 4 * | 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 |
Crystallization Reagents in Literatures
| ID | crystal ID | solution | reagent name | concentration (unit) | details |
| 1 | 1 | drop | hDHFR | 20 (mg/ml) | |
| 2 | 1 | drop | 25 (mM) | pH7.0 | |
| 3 | 1 | drop | EDTA | 0.1 (mM) | |
| 4 | 1 | drop | 3 (mM) | ||
| 5 | 1 | drop | NADPH | 2 (mM) | |
| 6 | 1 | drop | inhibitor | 2 (mM) | |
| 7 | 1 | reservoir | PEG4000 | 24-33 (%(w/v)) | |
| 8 | 1 | reservoir | 0.2 (M) | ||
| 9 | 1 | reservoir | Tris-HCl | 0.1 (M) | pH7.9-8.4 |
