8F5W
Dihydropyrimidine Dehydrogenase (DPD) C671S Mutant Soaked with Dihydrothymine and NADPH Quasi-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 | 2022-06-08 |
Detector | DECTRIS EIGER X 9M |
Wavelength(s) | 1.1272 |
Spacegroup name | P 1 21 1 |
Unit cell lengths | 81.735, 158.003, 162.404 |
Unit cell angles | 90.00, 96.06, 90.00 |
Refinement procedure
Resolution | 47.230 - 1.970 |
R-factor | 0.1806 |
Rwork | 0.179 |
R-free | 0.21700 |
Structure solution method | MOLECULAR REPLACEMENT |
Starting model (for MR) | 7m31 |
Data reduction software | xia2 |
Data scaling software | xia2 |
Phasing software | PHASER |
Refinement software | PHENIX (1.20.1_4487) |
Data quality characteristics
Overall | Inner shell | Outer shell | |
Low resolution limit [Å] | 47.230 | 52.290 | 2.010 |
High resolution limit [Å] | 1.970 | 5.350 | 1.970 |
Rmerge | 0.130 | 0.909 | |
Rmeas | 0.151 | 0.053 | 1.129 |
Rpim | 0.075 | 0.026 | 0.559 |
Number of reflections | 283435 | 14627 | 13645 |
<I/σ(I)> | 10 | 46.4 | 1.1 |
Completeness [%] | 98.6 | 100 | 95.2 |
Redundancy | 4 | 4.2 | 4 |
CC(1/2) | 0.994 | 0.633 |
Crystallization Conditions
crystal ID | method | pH | temperature | details |
1 | VAPOR DIFFUSION, HANGING DROP | 293 | DPD variant C671S (39.2 uM) in 25 mM HEPES, 2 mM DTT, 10% glycerol at pH 7.5 was mixed 1:1 with well solution containing 100 mM sodium citrate, 2 mM DTT, 19% PEG 6000 at pH 4.7 to yield a 6 uL drop. Crystallization was carried out in the dark to prevent photo-degradation of the somewhat dissociable FMN cofactor. Under these conditions DPD crystals appeared within 16 hours and were left to grow for additional 24 hours. Ligands were combined with the crystals under near anaerobic conditions as follows: before being placed in a Plas-Labs 830 series glove box, the well solution beneath selected crystallization drops were made anaerobic with the addition of 10 mM dithionite and resealed with the cover slide. Crystals trays were placed in the glove box that contained a Motic binocular microscope coupled to an Accuscope 1080p high-definition camera. The glove box was sealed and made quasi-anaerobic by flushing with high-purity nitrogen gas for approximately 10 minutes at which time fractional dioxygen was recorded as 0.1 %, by a Forensics Detectors oxygen meter. Atmospheric dioxygen was measured throughout the soaking procedure and was held <1%. C671S DPD crystals were soaked for a minimum of 20 minutes with DHT (R,S) (200 uM) and NADPH (100 uM), each dissolved in the following cryo-solution: 20 mM sodium citrate, 0.4 mM DTT, 20% PEG 6000, 20% PEG 400, pH 7.5. The crystals were then submerged in liquid nitrogen, removed from the anaerobic environment, and stored under liquid nitrogen. |