Fig. 3: CryoEM structure of the ATP-bound M854K MDA5-dsRNA filament.
a Domain architecture of human/mouse MDA5. CARD caspase recruitment domain; Hel1 and Hel2 first and second RecA-like helicase domains; Hel2i, Hel2 insert domain; P pincer domain; CTD C-terminal domain. The same color code is used in panels (b–e), Fig. 5a and Supplementary Fig. 4. b 3D density map of the ATP-bound M854K MDA5-dsRNA filament at 2.8 Å overall resolution, colored by domain as in (a), with RNA in magenta. An MDA5 protomer is outlined in dashed gray. c Overview of the atomic model of the ATP-bound M854K MDA5-dsRNA filament. The central protomer of three MDA5 protomers is outlined in light gray. ATP is shown in sphere representation. The region surrounding residue 854 is boxed. d, e Closeups of cryoEM density maps: the bound ATP, d, and the region surrounding residue 854, showing polar contacts formed by Lys854 with residues from the Hel1 and Hel2 domains, (e). Polar contacts formed by Lys854 are shown as dashed lines. f Closeup of the region surrounding residue 854 in the previously reported structure of the WT MDA5-dsRNA filament with AMPPNP bound14. g Relative ATPase activities of S491A/M854K, E813A/M854K, and S491A/E813A/M854K mutants, normalized to WT (100%) and M854K (0%). Error bars represent SEM between measurements, centered on the mean. (n = 3 independent samples; t = 15 min). ATPase assays were repeated in independent experiments three times with similar results. Source data are provided as a Source Data file.
