7M31

Dihydropyrimidine Dehydrogenase (DPD) C671S Mutant Soaked with Thymine 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	82.040, 159.560, 162.950
Unit cell angles	90.00, 95.75, 90.00

Refinement procedure

Resolution	36.150 - 1.690
R-factor	0.1779
Rwork	0.177
R-free	0.20010
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	1h7w
Data reduction software	xia2
Data scaling software	xia2
Phasing software	PHASER
Refinement software	PHENIX (1.19_4092)

Data quality characteristics

	Overall	Inner shell	Outer shell
Low resolution limit [Å]	45.350	45.350	1.730
High resolution limit [Å]	1.690	7.560	1.690
Rmerge	0.190	0.066	1.851
Rpim	0.096	0.033	0.944
Number of reflections	459508	5269	34249
<I/σ(I)>	6	20.3	0.9
Completeness [%]	99.0	98.8	99.8
Redundancy	4.7	4.7	4.8
CC(1/2)	0.992	0.993	0.350

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP		293	4.5 mg/mL (40 uM) of DPD variants, C671S 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%. C671S 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 thymine, 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.