6HAV

Crystal structure of [Fe]-hydrogenase (Hmd) from Methanococcus aeolicus in complex with FeGP and methenyl-tetrahydromethanopterin (close form A) at 1.06 A resolution

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	2017-02-17
Detector	DECTRIS PILATUS 6M
Wavelength(s)	1.00001
Spacegroup name	C 2 2 21
Unit cell lengths	66.648, 66.246, 167.599
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	45.240 - 1.060
R-factor	0.1187
Rwork	0.118
R-free	0.13360
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	6hac
RMSD bond length	0.011
RMSD bond angle	1.425
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]	46.980	1.120
High resolution limit [Å]	1.060	1.060
Rmerge	0.049	0.981
Rmeas	0.054	1.129
Rpim	0.021	0.542
Number of reflections	156432	18054
<I/σ(I)>	15.4	1.4
Completeness [%]	93.7	74.8
Redundancy	6.1	4
CC(1/2)	0.999	0.672

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 the 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 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 and 200 mM sodium thiocyanate with a ratio of protein mixture and crystallization reservoir of 2 ul / 2 ul drops spotted on a 24 well Junior Clover plate. The crystal was soaked for a couple of seconds in 20% w/v polyethylene glycol 3350, 20% v/v glycerol and 200 mM sodium thiocyanate before freezing in liquid nitrogen.