6HAE

Crystal structure of [Fe]-hydrogenase (Hmd) from Methanococcus aeolicus in complex with FeGP cofactor and methenyl-tetrahydromethanopterin (close form B)

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	2017-07-06
Detector	DECTRIS PILATUS 6M
Wavelength(s)	0.99992
Spacegroup name	P 21 21 2
Unit cell lengths	80.015, 156.480, 53.654
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	44.560 - 1.850
R-factor	0.163
Rwork	0.161
R-free	0.19200
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	6hac
RMSD bond length	0.010
RMSD bond angle	1.110
Data reduction software	XDS
Data scaling software	SCALA (3.3.22)
Phasing software	PHASER
Refinement software	BUSTER (2.10.3)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	44.560	1.950
High resolution limit [Å]	1.850	1.850
Rmerge	0.124	1.133
Rmeas	0.135	1.301
Rpim	0.055	0.627
Number of reflections	56771	6994
<I/σ(I)>	8.6	1.2
Completeness [%]	97.4	85
Redundancy	6	4.1
CC(1/2)	0.997	0.323

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	7	298	Crystallization of [Fe]-hydrogenase-methenyl-H4MPT+ complex was performed in an anaerobic tent with gas phase 100%N2 at room temperature under dark condition. The reconstituted [Fe]-hydrogenase holoenzyme (50 mg/ml) was mixed with 10-mM methenyl-H4MPT+, both of which contained 10-mM MOPS/KOH pH 7.0. The final concentrations of [Fe]-hydrogenase and methenyl-H4MPT+ were 24 mg/ml and 3 mM, respectively. After incubating the mixture in this tent under dark condition for 5 min, the enzyme solution was centrifuged using MiniSpin-plus (Eppendorf) at 8000 rpm for 5 min by using centrifugal filters made of polyvinylidene fluoride (PVDF, Millipore). The crystallization solution contained 20 % w/v polyethylene glycol 3350, 200 mM sodium thiocyanate and 3% w/v 1,5-diaminopentane dihydrochloride with a ratio of protein mixture and crystallization reservoir of 0.7 ul / 0.7 ul drops (in 96 well-plates).