5JC7

Crystal structure of chicken MDA5 with 5'p 24-mer dsRNA and ADP-Mg2+ at 2.75 A resolution.

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	ESRF BEAMLINE ID29
Synchrotron site	ESRF
Beamline	ID29
Temperature [K]	100
Detector technology	PIXEL
Collection date	2013-07-25
Detector	DECTRIS PILATUS3 6M
Wavelength(s)	0.9724
Spacegroup name	P 21 21 21
Unit cell lengths	99.750, 133.400, 138.440
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	50.000 - 2.750
R-factor	0.29224
Rwork	0.291
R-free	0.32199
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	5jc3
RMSD bond length	0.008
RMSD bond angle	1.212
Data reduction software	XDS
Data scaling software	XSCALE
Phasing software	PHASER
Refinement software	REFMAC (5.8.0131)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	50.000	2.820
High resolution limit [Å]	2.750	2.750
Rmerge	0.168	0.809
Number of reflections	48273
<I/σ(I)>	6.58	1.52
Completeness [%]	99.1	99.2
Redundancy	4.66	4.89

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	7.5	293	Directly after size exclusion chromatography chMDA5 deltaCARD-Q was mixed with 24-mer dsRNA in a 0.5:1 molar ratio and incubated for 30 minutes on ice. The complexes were concentrated to around 10 mg/ml and 2 mM AMPPNP and 200 mM NDSB211 (Dimethyl(2-hydroxyethyl)ammonium propane sulfonate) were added. chMDA5 delatCARD-Q:24bp dsRNA:AMPPNP was mixed with reservoir buffer (0.1 mM Hepes pH 7.5, 11-12% PEG 3350, 2-4% sucrose) in a 2:1 ratio. Crystals grew in three days at 20 degree and were harvested in cryo-protecting solution (0.1 mM Hepes pH 7.5, 25% 3350, 10% ethylene glycol) before flash freezing with liquid nitrogen