6HUZ

HmdII from Desulfurobacterium thermolithotrophum reconstituted with Fe-guanylylpyridinol (FeGP) cofactor and co-crystallized with methenyl-tetrahydrofolate form B

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	SOLEIL BEAMLINE PROXIMA 1
Synchrotron site	SOLEIL
Beamline	PROXIMA 1
Temperature [K]	100
Detector technology	PIXEL
Collection date	2018-06-07
Detector	DECTRIS PILATUS 6M
Wavelength(s)	0.97857
Spacegroup name	P 21 21 2
Unit cell lengths	65.200, 132.020, 49.160
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	46.390 - 1.850
R-factor	0.196
Rwork	0.195
R-free	0.22300
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	6huy
RMSD bond length	0.010
RMSD bond angle	1.140
Data reduction software	XDS
Data scaling software	SCALA (3.3.22)
Phasing software	PHASER (2.6.0)
Refinement software	BUSTER (2.10.3)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	49.160	1.950
High resolution limit [Å]	1.850	1.850
Rmerge	0.085	1.212
Rmeas	0.092	1.317
Rpim	0.036	0.511
Number of reflections	37060	5308
<I/σ(I)>	10.2	1.2
Completeness [%]	99.9	99.7
Redundancy	6.5	6.6
CC(1/2)	0.998	0.849

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	5.5	281	HmdII from Desulfurobacterium thermolithotrophum was reconstituted with the Fe-guanylylpyridinol cofactor from Methanothermobacter marburgensis and co-crystallized with methenyl-tetrahydrofolate using the sitting drop vapor diffusion method under N2/H2 (95%/5%) in red light condition. The reconstituted holoenzyme was mixed with methenyl-H4F+ at the final concentrations of 1.6 mM. Methenyl-tetrahydrofolate was dissolved in 10% DMSO, and the final concentration of DMSO in the protein solution was 1.6%. 0.7 ul of dHmdII at 21 mg/ml (reconstituted with FeGP and methenyl-H4F+) was spotted on a 96-well 2-drop MRC Crystallization Plates (Molecular Dimensions, Suffolk, UK) and 0.7 ul of reservoir solution was mixed. After one month, crystals appeared in 20% PEG 3000 (w/v) and 100 mM Sodium Citrate pH 5.5 (from the kit WIZARD, Jena Bioscience). The crystals were cryoprotected by a soak of few seconds in 20% PEG 3000 (w/v), 100 mM Tri-Sodium Citrate pH 5.5 and 20% glycerol before a flash freeze in liquid nitrogen