5JC7

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


X-RAY DIFFRACTION

Crystallization

Crystalization Experiments
IDMethodpHTemperatureDetails
1VAPOR DIFFUSION, SITTING DROP7.5293Directly 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
Crystal Properties
Matthews coefficientSolvent content
2.5752.18

Crystal Data

Unit Cell
Length ( Å )Angle ( ˚ )
a = 99.75α = 90
b = 133.4β = 90
c = 138.44γ = 90
Symmetry
Space GroupP 21 21 21

Diffraction

Diffraction Experiment
ID #Crystal IDScattering TypeData Collection TemperatureDetectorDetector TypeDetailsCollection DateMonochromatorProtocol
11x-ray100PIXELDECTRIS PILATUS3 6M2013-07-25MSINGLE WAVELENGTH
Radiation Source
ID #SourceTypeWavelength ListSynchrotron SiteBeamline
1SYNCHROTRONESRF BEAMLINE ID290.9724ESRFID29

Data Collection

Overall
ID #Resolution (High)Resolution (Low)Percent Possible (Observed)R Merge I (Observed)Net I Over Average Sigma (I)RedundancyNumber Reflections (All)Number Reflections (Observed)Observed Criterion Sigma (F)Observed Criterion Sigma (I)B (Isotropic) From Wilson Plot
12.755099.10.1686.584.6648273
Highest Resolution Shell
ID #Resolution (High)Resolution (Low)Percent Possible (All)Percent Possible (Observed)R Merge I (Observed)Mean I Over Sigma (Observed)RedundancyNumber Unique Reflections (All)
12.752.8299.20.8091.524.89

Refinement

Statistics
Diffraction IDStructure Solution MethodCross Validation methodStarting modelResolution (High)Resolution (Low)Number Reflections (Observed)Number Reflections (R-Free)Percent Reflections (Observed)R-Factor (Observed)R-WorkR-FreeR-Free Selection DetailsMean Isotropic B
X-RAY DIFFRACTIONMOLECULAR REPLACEMENTTHROUGHOUT5JC32.755045840243399.080.292240.290640.32199RANDOM69.662
Temperature Factor Modeling
Anisotropic B[1][1]Anisotropic B[1][2]Anisotropic B[1][3]Anisotropic B[2][2]Anisotropic B[2][3]Anisotropic B[3][3]
7.42.21-9.61
RMS Deviations
KeyRefinement Restraint Deviation
r_dihedral_angle_2_deg36.299
r_dihedral_angle_3_deg16.126
r_dihedral_angle_4_deg14.386
r_long_range_B_refined7.322
r_long_range_B_other7.322
r_dihedral_angle_1_deg6.279
r_mcangle_it5.072
r_mcangle_other5.072
r_scangle_other4.224
r_mcbond_it2.972
RMS Deviations
KeyRefinement Restraint Deviation
r_dihedral_angle_2_deg36.299
r_dihedral_angle_3_deg16.126
r_dihedral_angle_4_deg14.386
r_long_range_B_refined7.322
r_long_range_B_other7.322
r_dihedral_angle_1_deg6.279
r_mcangle_it5.072
r_mcangle_other5.072
r_scangle_other4.224
r_mcbond_it2.972
r_mcbond_other2.97
r_scbond_it2.409
r_scbond_other2.409
r_angle_refined_deg1.212
r_angle_other_deg0.941
r_chiral_restr0.066
r_bond_refined_d0.008
r_gen_planes_refined0.004
r_bond_other_d0.002
r_gen_planes_other0.002
r_nbd_refined
r_nbd_other
r_nbtor_refined
r_nbtor_other
r_xyhbond_nbd_refined
r_xyhbond_nbd_other
r_metal_ion_refined
r_metal_ion_other
r_symmetry_vdw_refined
r_symmetry_vdw_other
r_symmetry_hbond_refined
r_symmetry_hbond_other
r_symmetry_metal_ion_refined
r_symmetry_metal_ion_other
r_scangle_it
r_rigid_bond_restr
r_sphericity_free
r_sphericity_bonded
Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen AtomsNumber
Protein Atoms10537
Nucleic Acid Atoms1021
Solvent Atoms
Heterogen Atoms58

Software

Software
Software NamePurpose
REFMACrefinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing