1KMV
HUMAN 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
Experimental procedure
Experimental method | SINGLE WAVELENGTH |
Source type | SYNCHROTRON |
Source details | NSLS BEAMLINE X25 |
Synchrotron site | NSLS |
Beamline | X25 |
Temperature [K] | 100 |
Detector technology | CCD |
Collection date | 1998-06-18 |
Detector | BRANDEIS - B4 |
Spacegroup name | P 21 21 21 |
Unit cell lengths | 41.300, 54.990, 82.680 |
Unit cell angles | 90.00, 90.00, 90.00 |
Refinement procedure
Resolution | 20.000 - 1.050 |
Rwork | 0.130 |
R-free | 0.18100 * |
Structure solution method | MOLECULAR REPLACEMENT |
Starting model (for MR) | UNPUBLISHED HUMAN DHFR STRUCTURE. |
RMSD bond length | 0.017 |
RMSD bond angle | 2.270 * |
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 | 1.090 |
High resolution limit [Å] | 1.050 | 1.050 |
Rmerge | 0.053 | 0.231 |
Total number of observations | 312507 * | |
Number of reflections | 86349 | |
<I/σ(I)> | 9.6 | 2.8 |
Completeness [%] | 97.5 | 83.8 |
Redundancy | 3.6 | 2.1 |
Crystallization Conditions
crystal ID | method | pH | temperature | details |
1 | VAPOR DIFFUSION, HANGING DROP | 7 * | 4 * | 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 |
Crystallization Reagents in Literatures
ID | crystal ID | solution | reagent name | concentration (unit) | details |
1 | 1 | drop | hDHFR | 20 (mg/ml) | |
10 | 1 | reservoir | glycerol | 5 (%(v/v)) | for hDHFR/NADPH/SRI-9662 ternary complex |
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 |