Fig. 2. Cryo-EM structure and mutagenesis of the CARD8-CT filament.

a, b Overall structure of the CARD8-CT inflammasome, with a resolved CARD8^CARD filament. The cryo-EM density (a) and atomic model (b) are colored by subunit. c Illustration of classical CARD filament interfaces: type I, II, and III. d Detailed CARD–CARD interactions in the CARD8-CT filament structure colored as in c. e Negative stain electron microscopy snapshots of CARD8^CARD wild-type (WT) and mutants that abolish filament formation in vitro. Filaments were formed at 15 µM (shown here) and 30 µM. Micrographs are representative of >3 fields of view for each sample. f Inflammasome assay of CARD8-CT-mCherry mutants transiently expressed in HEK293T cells that stably express caspase-1 and GSDMD. Filament-deficient mutants abolish inflammasome activity as measured by LDH release (top) and western blot (bottom) for activated inflammasome components. P-values are compared to empty vector (EV) by two-sided Student’s t-test. Exact P-values are provided in the Source Data File. Data are means ± SEM of n = 3 biological replicates. g Confocal imaging of HEK293T cells transiently expressing CARD8^UPA-CARD-mCherry WT and mutants. Filament-deficient mutants abolish filament formation in cells. Experiments were performed with two biological replicates. Scale bar 5 µm. In e–g, mutations are labeled in the colors of the interface types as defined in c.