9R6Z

Cubic state of the F420-reducing hydrogenase from Methanothermococcus thermolithotrophicus

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	ESRF BEAMLINE BM30A
Synchrotron site	ESRF
Beamline	BM30A
Temperature [K]	100
Detector technology	CCD
Collection date	2016-09-23
Detector	ADSC QUANTUM 315r
Wavelength(s)	1.3871
Spacegroup name	P 21 3
Unit cell lengths	282.072, 282.072, 282.072
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	31.340 - 2.850
R-factor	0.1837
Rwork	0.182
R-free	0.21010
Structure solution method	MOLECULAR REPLACEMENT
RMSD bond length	0.006
RMSD bond angle	0.878
Data reduction software	XDS
Data scaling software	SCALA
Phasing software	PHASER
Refinement software	PHENIX ((1.20.1_4487: ???))

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	49.100	3.000
High resolution limit [Å]	2.850	2.850
Rmerge	0.217	1.497
Rmeas	0.233	1.605
Rpim	0.085	0.572
Number of reflections	173025	25112
<I/σ(I)>	6	1.2
Completeness [%]	100.0	100
Redundancy	7.6	7.8
CC(1/2)	0.988	0.433

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	7.5	293.15	The enzyme used for crystallisation was concentrated at 16 mg/ml in a buffer containing 25 mM Tris/HCl pH 7.6, 10% (v/v) glycerol, 2 mM dithiothreitol and 2 mM FAD. Crystals were obtained anaerobically in a Coy tent filled with a N2/H2 (97:3 %) atmosphere by initial screening at 20 degree Celsius using the sitting-drop method on 96-well MRC two-drop crystallisation plates in polystyrene (SWISSCI) containing 90 ul of crystallisation solution in the reservoir. The crystallisation solution was 1.5 M LiSO4 and 100 mM HEPES pH 7.5. Prior to freezing, the crystal was soaked in the crystallisation solution supplemented with 30% (v/v) glycerol.