Structural Analysis of FLAG-FADD Complexes

Negative stain TEM data were collected on well-dried specimens using a Talos electron microscope (FEI), operated at 200 kV acceleration. Images were recorded using the EPU system (Thermo Fisher Scientific, MA USA) at room temperature using a 4096 × 4096 pixel CMOS camera Ceta16M (FEI), at a magnification of x 57,000 (pixel size 1.83 Å), 1 s exposure per micrograph. FLAG-FADD complexes were manually picked from 620 micrographs yielding a dataset of 3200 particles, as summarized in Supplementary Table 1. The single-particle analysis was then performed using RELION^{27 (link),52 (link)} and initial models generated and refined in CryoSPARC^{53 (link)}, as summarized in Supplementary Figs. 4, and 7.