5OK4

Crystal structure of native [Fe]-hydrogenase Hmd from Methanothermobacter marburgensis inactivated by O2.

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	SLS BEAMLINE X10SA
Synchrotron site	SLS
Beamline	X10SA
Temperature [K]	100
Detector technology	PIXEL
Collection date	2016-11-14
Detector	DECTRIS PILATUS 6M
Wavelength(s)	1.00005
Spacegroup name	H 3 2
Unit cell lengths	127.418, 127.418, 141.211
Unit cell angles	90.00, 90.00, 120.00

Refinement procedure

Resolution	43.474 - 1.290
R-factor	0.128
Rwork	0.127
R-free	0.14710
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	2jjf
RMSD bond length	0.010
RMSD bond angle	1.194
Data reduction software	XDS
Data scaling software	SCALA (3.3.22)
Phasing software	PHASER
Refinement software	PHENIX ((1.10.1_2155: ???))

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	47.070	1.360
High resolution limit [Å]	1.290	1.290
Rmerge	0.048	1.128
Rpim	0.018	0.423
Number of reflections	110077	15981
<I/σ(I)>	19.6	1.9
Completeness [%]	100.0	100
Redundancy	8.2	8
CC(1/2)	1.000	0.638

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	7	281.15	1-ml mixture containing 120 mM potassium phosphate pH 6.0, 1 mM EDTA, 26 uM (1 mg/ml) [Fe]-hydrogenase and 26 uM methylene-H4MPT was incubated in 5-ml amber vials with rubber stoppers under a gas mixture of N2/O2/H2 (70%/20%/10%) at 40 degree Celsius for 1 h. The inactivated enzyme was concentrated to 25 mg/ml using a 30 kDa centrifugal filter (Millipore). 0.7-ul of 25 mg/ml O2-inactivated [Fe]-hydrogenase was mixed with 0.7 ul of reservoir solution of 2 M (NH4)2SO4, 100 mM Tris/HCl, pH 7.0, and 200 mM LiSO4. The best diffracting crystal was obtained in one month. Prior to freezing the crystal was soaked for 3 seconds in 2 M (NH4)2SO4, 100 mM Tris/HCl, pH 7.0, 200 mM LiSO4 and 30% glycerol v/v.