2C2T
Human Dihydrofolate Reductase Complexed With NADPH and 2,4-Diamino-5-((7,8-dicarbaundecaboran-7-yl)methyl)-6-methylpyrimidine, a novel boron containing, nonclassical Antifolate
Experimental procedure
Experimental method | SINGLE WAVELENGTH |
Source type | SYNCHROTRON |
Source details | SSRL BEAMLINE BL9-1 |
Synchrotron site | SSRL |
Beamline | BL9-1 |
Temperature [K] | 100 |
Detector technology | IMAGE PLATE |
Collection date | 1999-03-03 |
Detector | MARRESEARCH |
Spacegroup name | C 2 2 21 |
Unit cell lengths | 87.628, 94.354, 96.032 |
Unit cell angles | 90.00, 90.00, 90.00 |
Refinement procedure
Resolution | 20.000 - 1.500 |
R-factor | 0.16 |
Rwork | 0.158 |
R-free | 0.21300 |
Structure solution method | MOLECULAR REPLACEMENT |
Starting model (for MR) | UNPUBLISHED HUMAN DHFR STRUCTURE |
RMSD bond length | 0.012 |
RMSD bond angle | 1.451 |
Data reduction software | MOSFLM |
Data scaling software | SCALA |
Phasing software | AMoRE |
Refinement software | REFMAC (5.2.0019) |
Data quality characteristics
Overall | Outer shell | |
Low resolution limit [Å] | 35.000 | 1.540 |
High resolution limit [Å] | 1.500 | 1.500 |
Rmerge | 0.060 | 0.450 |
Number of reflections | 62594 | |
<I/σ(I)> | 8.4 | 1.7 |
Completeness [%] | 98.4 | 90.1 |
Redundancy | 2.9 | 2.1 |
Crystallization Conditions
crystal ID | method | pH | temperature | details |
1 | VAPOR DIFFUSION, HANGING DROP | 8 | 277 | THE TERNARY COMPLEX OF DHFR WITH NADPH AND 39B 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. NOTE THAT THE INHIBITOR IS ACTUALLY A RACEMIC MIXTURE OF 39B AND ITS ENANTIOMER, HERE CALLED 39E. IT IS THE RACEMATE THAT IS BOUND IN THE CRYSTAL, REPRESENTED IN THE COORDINATES AS A 1:1 STATISTICALLY-DISORDERED MIXTURE OF 39B AND 39E. |