9OER

HalA with lysine, Fe(II), chloride, and a peroxyhemiketal intermediate

This is a non-PDB format compatible entry.

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	ALS BEAMLINE 8.3.1
Synchrotron site	ALS
Beamline	8.3.1
Temperature [K]	90
Detector technology	PIXEL
Collection date	2022-12-22
Detector	DECTRIS PILATUS3 S 6M
Wavelength(s)	1.11583
Spacegroup name	H 3
Unit cell lengths	147.300, 147.300, 287.870
Unit cell angles	90.00, 90.00, 120.00

Refinement procedure

Resolution	95.960 - 1.720
R-factor	0.1784
Rwork	0.168
R-free	0.20120
Structure solution method	MOLECULAR REPLACEMENT
RMSD bond length	0.011
RMSD bond angle	0.678
Data reduction software	XDS (June 1, 2017)
Data scaling software	Aimless (0.7.7)
Phasing software	PHASER (2.8.3)
Refinement software	PHENIX (1.21.2_5419)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	95.960	1.781
High resolution limit [Å]	1.720	1.720
Rmerge	0.181	2.376
Rmeas	0.186	2.422
Rpim	0.041	0.464
Number of reflections	246753	24600
<I/σ(I)>	19.5	1.84
Completeness [%]	99.7	99.13
Redundancy	38.94	26.67
CC(1/2)	0.996	0.657

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP	7	298	HalA crystals were obtained by the hanging drop vapor diffusion method by combining equal volumes of protein solution (HalA (5 mg/ml), lysine (50 mM, pH 7), and aKG (20 mM, pH 7)) and reservoir solution (potassium phosphate monobasic (40 mM), 20% (w/v) PEG 8000, 15% (v/v) glycerol). Crystals grew in three days and were transferred to an Eppendorf tube containing 250 uL of reservoir solution and vortexed for 30 seconds with 10 x 1 uM diameter zirconia/silica beads to produce a micro-seed solution. The seed solution was stored at -80C for future use. Subsequent crystals were prepared by micro-seeding equal volumes of protein solution (HalA (5 mg/ml), lysine (50 mM, pH 7), and aKG (20 mM, pH 7)) and reservoir solution (potassium phosphate monobasic (40 mM), 20% (w/v) PEG 8000, 15% (v/v) glycerol). Crystals grew after two weeks inside a Coy anaerobic chamber. Crystals were soaked with (NH4)2Fe(SO4)2 6H2O (1.42 mM) while NO accumulated in the well. To introduce NO, diethylamine NONOate was prepared by dissolving solid in 10 mM NaOH. The stock concentration was determined based on the published extinction coefficient of e250 = 6,500 M-1 cm-1 using a Nanodrop. NONOate was added to a final concentration of 26 mM in each well along with 150 mM HEPES pH 7.5 to trigger NONOate decomposition. The wells were quickly re-sealed and NO was allowed to accumulate for approximately 1 hour, after which the crystals were flash frozen in liquid nitrogen