8A8O

PAPS reductase from Methanothermococcus thermolithotrophicus refined to 1.45 A

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	SLS BEAMLINE X06DA
Synchrotron site	SLS
Beamline	X06DA
Temperature [K]	100
Detector technology	PIXEL
Collection date	2021-04-21
Detector	DECTRIS PILATUS 2M-F
Wavelength(s)	0.97625
Spacegroup name	P 1 21 1
Unit cell lengths	63.714, 123.650, 88.842
Unit cell angles	90.00, 104.44, 90.00

Refinement procedure

Resolution	52.040 - 1.450
R-factor	0.1527
Rwork	0.151
R-free	0.17800
Structure solution method	AB INITIO PHASING
Data reduction software	autoPROC
Data scaling software	autoPROC
Phasing software	CRANK2
Refinement software	PHENIX (1.19.2_4158)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	86.040	1.640
High resolution limit [Å]	1.450	1.450
Rmerge	0.079	1.056
Rmeas	0.086	1.139
Rpim	0.033	0.425
Number of reflections	146296	7316
<I/σ(I)>	11.9	1.7
Completeness [%]	95.0	73.8
Redundancy	6.8	7.1
CC(1/2)	0.997	0.567

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	7	293	Purified PAPS reductase from Methanothermococcus thermolithotrophicus at a concentration of 20 mg.ml-1 was anaerobically crystallized inside a Coy tent (N2/H2 at a 96:4 ratio) under yellow light condition. 0.7 ul of PAPSR in 25 mM Tris/HCl pH 7.6, 2 mM dithiothreitol, 0.5 mM FAD, and 10 % v/v glycerol was mixed with 0.7 ul reservoir solution. The drops were spotted in a 96-Well MRC 2-drop crystallization plates in polystyrene (SWISSCI) containing 90 ul of crystallization solution in the reservoir. Crystals appeared after a few days in the following crystallization condition: 35 % v/v 2-Methyl-2,4-pentanediol, 100 mM Tris pH 7.0 and 200 mM NaCl. Prior to transfer to liquid nitrogen, the crystal was soaked in 10 mM disodium 3'-Phosphoadenosine 5'-phosphate for 7 min.