6HUX

HmdII from Methanocaldococcus jannaschii reconstitued with Fe-guanylylpyridinol (FeGP) cofactor and co-crystallized with methenyl-tetrahydromethanopterin at 2.5 A resolution

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	ESRF BEAMLINE BM30A
Synchrotron site	ESRF
Beamline	BM30A
Temperature [K]	100
Detector technology	CCD
Collection date	2017-04-22
Detector	ADSC QUANTUM 315r
Wavelength(s)	0.9799
Spacegroup name	H 3 2
Unit cell lengths	124.281, 124.281, 150.086
Unit cell angles	90.00, 90.00, 120.00

Refinement procedure

Resolution	38.970 - 2.500
R-factor	0.168
Rwork	0.165
R-free	0.21900
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	4yt4
RMSD bond length	0.008
RMSD bond angle	0.990
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]	43.730	2.640
High resolution limit [Å]	2.500	2.500
Rmerge	0.122	1.341
Rmeas	0.129	1.415
Rpim	0.041	0.447
Number of reflections	15610	2224
<I/σ(I)>	13.1	2.2
Completeness [%]	100.0	99.9
Redundancy	9.8	9.9
CC(1/2)	0.997	0.686

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	5.5	281	HmdII from Methanocaldococcus jannaschii reconstituted with Fe-guanylylpyridinol cofactor was cocrystallized with methenyl-tetrahydromethanopterin using the sitting drop vapor diffusion method under N2/H2 (95%/5%) in red light condition. 20 mg/ml of reconstituted enzyme in 25 mM Tris pH 7.5, 5% glycerol, 150 mM NaCl, 2 mM DTT and 3 mM methenyl-tetrahydromethanopterin was spotted on a 96-well 2-drop MRC Crystallization Plates (Molecular Dimensions, Suffolk, UK) with a ratio of 0.7 ul of protein and 0.7 ul of reservoir solution. After several weeks, crystals appeared in 2 M LiSO4, 100 mM Sodium acetate pH 5.5, 100 mM MgSO4 and 5% v/v PEG 400. jHmdII crystal was cryoprotected in its mother liquor supplemented with 30% ethylene glycol before freezing in liquid nitrogen.