6HUY

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

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	2018-04-15
Detector	DECTRIS PILATUS 6M
Wavelength(s)	1.00000
Spacegroup name	P 21 21 21
Unit cell lengths	99.769, 131.829, 168.263
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	24.983 - 2.250
R-factor	0.1881
Rwork	0.187
R-free	0.21220
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	4yt4
RMSD bond length	0.007
RMSD bond angle	0.879
Data reduction software	XDS
Data scaling software	SCALA (3.3.22)
Phasing software	PHASER (2.6.0)
Refinement software	PHENIX ((1.10.1_2155: ???))

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	48.890	2.370
High resolution limit [Å]	2.250	2.250
Rmerge	0.096	1.421
Rmeas	0.108	1.595
Rpim	0.049	0.714
Number of reflections	105630	15270
<I/σ(I)>	9.7
Completeness [%]	99.9	100
Redundancy	4.8	4.9
CC(1/2)	0.998	0.623

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	5.5	281	dHmdII reconstituted with the Fe-guanylylpyridinol cofactor from Methanothermobacter marburgensis was cocrystallized with methenyl-tetrahydrofolate using the sitting drop vapor diffusion method at 281 K under N2/H2 (95%/5%) in red light condition. The reconstituted dHmdII holoenzyme was mixed with methenyl-tetrahydrofolate 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 (with Fe-guanylylpryridinol and methenyl-tetrahydrofolate) 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 10 days, the crystals appeared in 20% PEG 3000 (w/v), 100 mM Tri-Sodium Citrate pH 5.5 (from the kit Wizard, Jean Bioscience). 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.