5JC3

Crystal structure of chicken MDA5 with 5'p 10-mer dsRNA and ADP-Mg2+ at 2.6 A resolution (monoclinic form, twinned).

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	2014-03-02
Detector	DECTRIS PILATUS3 6M
Wavelength(s)	0.9724
Spacegroup name	P 1 21 1
Unit cell lengths	70.160, 138.700, 100.430
Unit cell angles	90.00, 109.47, 90.00

Refinement procedure

Resolution	50.000 - 2.600
R-factor	0.27205
Rwork	0.271
R-free	0.28962
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	4gl2
RMSD bond length	0.007
RMSD bond angle	1.113
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	2.690
High resolution limit [Å]	2.600	2.600
Rmerge	0.096	0.896
Number of reflections	55300
<I/σ(I)>	9.83	1.73
Completeness [%]	99.3	99.6
Redundancy	3.52	3.63

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, SITTING DROP	7	293	Directly after size exclusion chromatography chMDA5 deltaCARD-Q was mixed with 10 bp dsRNA in a 1:1 molar ratio and incubated for 30 minutes on ice. The complex was concentrated using an Amicon Ultra concentrator to around 10 mg/ml and 2 mM AMPPNP (adenosine 5prime-(beta,gamma-imido)triphosphate lithium salt hydrate) was added. Sample and reservoir buffer (0.025 M Bis-Tris pH 6.5, 0.075 M succinic acid pH 7.0, 12-14% PEG 3350, 2% sucrose) were mixed in a 2:1 ratio. Three hours after setup, cover glasses with drops were transferred from a reservoir containing 12-14% PEG 3350 to one containing 8% PEG 3350. Crystals grew in one week at 20 C and were harvested in cryo-protectant solution (0.025 M Bis-Tris pH 6.5, 0.075 M succinic acid pH 7.0, 25% 3350, 10% ethylene glycol) before flash freezing with liquid nitrogen.