7M32
Dihydropyrimidine Dehydrogenase (DPD) C671A Mutant Soaked with Uracil and NADPH Anaerobically
Experimental procedure
Experimental method | SINGLE WAVELENGTH |
Source type | SYNCHROTRON |
Source details | APS BEAMLINE 21-ID-D |
Synchrotron site | APS |
Beamline | 21-ID-D |
Temperature [K] | 100 |
Detector technology | PIXEL |
Collection date | 2020-12-09 |
Detector | DECTRIS EIGER X 9M |
Wavelength(s) | 1.1272 |
Spacegroup name | P 1 21 1 |
Unit cell lengths | 81.870, 158.730, 162.700 |
Unit cell angles | 90.00, 95.89, 90.00 |
Refinement procedure
Resolution | 47.260 - 1.820 |
R-factor | 0.1706 |
Rwork | 0.169 |
R-free | 0.20190 |
Structure solution method | MOLECULAR REPLACEMENT |
Starting model (for MR) | 1h7w |
Data reduction software | autoPROC |
Data scaling software | Aimless (0.7.4) |
Phasing software | PHASER |
Refinement software | PHENIX (1.19_4092) |
Data quality characteristics
Overall | Inner shell | Outer shell | |
Low resolution limit [Å] | 51.690 | 51.690 | 1.870 |
High resolution limit [Å] | 1.820 | 8.140 | 1.820 |
Rmerge | 0.147 | 0.041 | 1.946 |
Rmeas | 0.165 | 0.046 | 2.179 |
Rpim | 0.074 | 0.020 | 0.969 |
Total number of observations | 1705022 | 19158 | 127495 |
Number of reflections | 354924 | 4165 | 26146 |
<I/σ(I)> | 7.4 | 27.5 | 0.9 |
Completeness [%] | 96.5 | 98.6 | 96.2 |
Redundancy | 4.8 | 4.6 | 4.9 |
CC(1/2) | 0.996 | 0.998 | 0.341 |
Crystallization Conditions
crystal ID | method | pH | temperature | details |
1 | VAPOR DIFFUSION, HANGING DROP | 293 | ~4.5 mg/mL (~40 uM) of C671A DPD in 25 mM HEPES, 2 mM DTT, 10% glycerol at pH 7.5 was prepared and diluted 1:1 with well solution containing 100 mM sodium citrate, 2 mM DTT, 15% PEG 6000 at pH 4.5 to 4.9. Diffraction quality crystals with elongated morphology were obtained by the hanging-drop vapor diffusion method. Incubation was carried out in the dark to eliminate photo-degradation the quasi-labile FMN cofactor. Crystals appeared after 16 hours as both single elongated rectangular hexahedron forms (200 x 50 x 50) or urchin-like clusters. Only the single crystal form were harvested and these were placed in a Plas-Labs 830 series glove box into which a Motic binocular microscope coupled to an Accuscope 1080 P HD camera was placed. Before being placed in the glove box, the well solutions of the selected crystals were made anaerobic with the addition of 10 mM dithionite and resealed with the cover slide. The glove box was then made anaerobic by flushing with pure nitrogen for approximately 10 minutes at which time the fractional dioxygen was 0.1 %, as indicated by a Forensics Detectors oxygen meter. Atmospheric dioxygen was measured throughout the soaking procedure and was held below 1%. C671A DPD crystals were soaked for a minimum of 20 minutes in 25 mM HEPES, 100 mM sodium citrate, 2 mM DTT, 100 uM NADPH, 100 uM uracil, 20% PEG 6000, 20% PEG 400, pH 7.5 prior to submersion in liquid nitrogen. Frozen crystals were then removed from the anaerobic environment and stored under liquid nitrogen. |