Table 1.
Cryo-EM Data Collection, Structure Determination, Model Building, and Refinement Parameters and Statistics
| Mouse MDA5 Bound to Phi6 dsRNA 1 mM AMPPNP | MDA5-dsRNA, 2.5 mM AMPPNP | MDA5-dsRNA, 10 mM ATP | MDA5-dsRNA, 2 mM ADP-AlF4 | MDA5-dsRNA, No Nucleotide | ||||
|---|---|---|---|---|---|---|---|---|
| Data collection and processing | ||||||||
| Microscope | FEI Titan Krios | FEI Titan Krios | FEI Titan Krios | FEI Titan Krios | FEI Titan Krios | |||
| Voltage (kV) | 300 | 300 | 300 | 300 | 300 | |||
| Electron exposure (electrons Å−2) | 29.85 | 29.33 | 30.24 | 29.85/30.50 | 27.0 | |||
| Exposure per frame (electrons Å−2) | 0.398 | 0.391 | 0.403 | 0.398/0.272 | 1.00 | |||
| Defocus range (μm) | −1.8 to −2.7 | −1.8 to −2.7 | −1.7 to −3.1 | −1.8 to −2.7 | −1.8 to −2.7 | |||
| Pixel size (Å) | 1.07 | 1.07 | 1.07 | 1.07/1.085 | 1.04 | |||
| No. of initial segment images | 367,549 | 207,000 | 526,596 | 234,835 | 137,601 | |||
| 3D class average | Twist74 | Twist87 | Twist91 | Twist77-2.5 | Twist72-10 | ADP-AlF4 | Tw89-NoNt | Tw93-NoNt |
| No. of final segment images | 33,138 | 60,079 | 40,265 | 28,663 | 100,482 | 31,556 | 26,527 | 19,111 |
| Map resolution range (Å) | 240–3.68 | 240–3.93 | 240–3.93 | 240–4.06 | 240–3.87 | 240–4.06 | 240–4.02 | 240–4.16 |
| Fourier shell correlation (FSC) threshold for resolution limit | 0.143 | 0.143 | 0.143 | 0.143 | 0.143 | 0.143 | 0.143 | 0.143 |
| Max. local resolution range | 24.0–3.42 | 20.0–3.63 | 20.0–3.69 | 26.6–3.63 | 21.8–3.58 | 26.6–3.99 | 50.0–3.22 | 50.06–3.89 |
| Model refinement | ||||||||
| Map sharpening B factor (Å2) | −175 | −165 | −175 | −125 | −175 | −175 | −150 | −175 |
| Helical symmetry imposed | ||||||||
| Twist (°) | 74.302 | 87.369 | 90.921 | 76.776 | 72.817 | 87.832 | 89.000 | 93.060 |
| Rise (Å) | 42.844 | 44.510 | 44.970 | 43.106 | 43.062 | 46.511 | 44.242 | 44.366 |
| Mask correlation coefficient | 0.767 | 0.767 | 0.762 | 0.795 | 0.818 | 0.798 | 0.801 | 0.775 |
| Model composition | ||||||||
| No. of non-hydrogen atoms | 6,209 | 5,894 | 5,907 | 6,209 | 6,268 | 6,296 | 5,955 | 5,997 |
| Protein residues | 682 | 645 | 648 | 682 | 689 | 685 | 660 | 667 |
| RNA nucleotides | 28 | 30 | 30 | 28 | 28 | 30 | 30 | 30 |
| Ligand | AMPPMP | – | – | AMPPMP | ATP | ADP-AlF4 | – | – |
| Ions (Zn2+ or Zn2+ and Mg2+) | 1 | 1 | 1 | 1 | 2 | 1 | 1 | 1 |
| RMSDs | ||||||||
| Bond lengths (Å) | 0.012 | 0.006 | 0.010 | 0.011 | 0.007 | 0.006 | 0.007 | 0.008 |
| Bond angles (°) | 1.093 | 1.000 | 1.242 | 1.050 | 0.896 | 0.938 | 0.820 | 0.872 |
| Planarity (Å) | 0.006 | 0.004 | 0.005 | 0.006 | 0.004 | 0.004 | 0.005 | 0.006 |
| B-factors and ADPs | ||||||||
| Minimum | 30.0 | 54.4 | 51.0 | 102 | 75.6 | 57.9 | 76.1 | 36.8 |
| Maximum | 100 | 170 | 156 | 263 | 187 | 159 | 198 | 145 |
| Mean | 56.0 | 103 | 100 | 167 | 115 | 95.4 | 136 | 78.8 |
| Validation | ||||||||
| MolProbity overall score | 1.71 | 1.79 | 1.83 | 1.77 | 1.65 | 1.74 | 1.71 | 1.76 |
| MolProbity all-atom clash score | 3.52 | 3.84 | 4.37 | 4.00 | 3.09 | 4.03 | 3.87 | 4.19 |
| Rotamer outliers (%) | 0 | 0 | 0 | 0 | 0.11 | 0 | 0.18 | 0.17 |
| Ramachandran plot | ||||||||
| Favored (%) | 89.4 | 87.3 | 87.3 | 88.8 | 90.1 | 90.0 | 90.5 | 89.5 |
| Allowed (%) | 10.3 | 12.7 | 12.7 | 10.9 | 9.7 | 10.0 | 9.5 | 10.3 |
| Outliers (%) | 0.3 | 0 | 0 | 0.3 | 0.2 | 0 | 0 | 0.2 |
| PDB codes | PDB: 6G19 | PDB: 6G1S | PDB: 6G1X | PDB: 6GJZ | PDB: 6GKM | PDB: 6GKH | PDB: 6H61 | PDB: 6H66 |
| EMDB codes | EMD-4338 | EMD-4340 | EMD-4341 | EMD-0012 | EMD-0024 | EMD-0023 | EMD-0143 | EMD-0145 |
| EMPIAR codes | 10213 | 10213 | 10213 | 10209 | 10208 | 10211 | 10210 | 10210 |