Summary Double-stranded RNA (dsRNA) is a potent proinflammatory signature of viral infection. Long cytosolic dsRNA is recognized by MDA5. The cooperative assembly of MDA5 into helical filaments on dsRNA nucleates the assembly of a multiprotein type-I-interferon signaling platform. Here, we determined cryoEM structures of MDA5-dsRNA filaments with different helical twists and bound nucleotide analogs, at resolutions sufficient to build and refine atomic models. The structures identify the filament forming interfaces, which encode the dsRNA binding cooperativity and length specificity of MDA5. The predominantly hydrophobic interface contacts confer flexibility, reflected in the variable helical twist within filaments. Mutation of filament-forming residues can result in loss or gain of signaling activity. Each MDA5 molecule spans 14 or 15 RNA base pairs, depending on the twist. Variations in twist also correlate with variations in the occupancy and type of nucleotide in the active site, providing insights on how ATP hydrolysis contributes to MDA5-dsRNA recognition. eTOC Structures of MDA5 bound to double-stranded RNA reveal a flexible, predominantly hydrophobic filament forming interface. The filaments have variable helical twist. Structures determined with ATP and transition state analogs show how the ATPase cycle is coupled to changes in helical twist, the mode of RNA binding and the length of the RNA footprint of MDA5. Highlights <jats:list list-type="bullet">jats:list-item CryoEM structures of MDA5-dsRNA filaments determined for three catalytic states jats:list-item Filament forming interfaces are flexible and predominantly hydrophobic jats:list-item Mutation of filament-forming residues can cause loss or gain of IFN-β signaling jats:list-item ATPase cycle is coupled to changes in filament twist and size of the RNA footprint