8F61

Dihydropyrimidine Dehydrogenase (DPD) C671S Mutant Soaked with Dihydrothymine 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	81.784, 157.667, 162.564
Unit cell angles	90.00, 95.93, 90.00

Refinement procedure

Resolution	161.690 - 2.140
R-factor	0.1891
Rwork	0.187
R-free	0.22980
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	7m31
RMSD bond length	0.003
RMSD bond angle	0.644
Data reduction software	xia2
Data scaling software	xia2
Phasing software	PHASER
Refinement software	PHENIX (1.20.1_4487)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	161.690	2.450
High resolution limit [Å]	2.140	2.140
Rmerge	0.172	0.762
Rmeas	0.203	0.893
Rpim	0.103	0.459
Number of reflections	118212	21096
<I/σ(I)>	7.2	2
Completeness [%]	89.3	55.9
Redundancy	3.7	3.6
CC(1/2)	0.989	0.485

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), 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.