Falcon 2 direct electron detector

Samples were diluted to a final concentration of 20–50 μg ml⁻¹ (20 mM HEPES (pH 8.0), 40 mM KOAc, 5 mM MgCl₂, 0.1% trehalose, 2 mM DTT and 0.01% NP-40), and 3 μl of aliquots was applied to freshly glow-discharged Quantifoil R2/1 grids coated with a second layer of thin carbon film. The grids were blotted for 3–4 s at 4 °C in 100% humility, and then plunged into liquid ethane using an FEI Vitrobot (FEI Company). Frozen grids were stored in liquid nitrogen. The grids were first loaded into a Gatan 626 cryo-holder and transferred to an FEI Tecnai TF20 electron microscope to check the quality of the sample vitrification. Then, the grids were transferred to Titan Krios equipped with a field emission source and were operated at 300 kV. Images were recorded on a Falcon 2 direct electron detector at a nominal magnification of × 59,000 with a defocus range of 2–4 μm, resulting in a calibrated sampling of 1.42 Å per pixel. The total accumulated dose rate was set to be 35 e² per Ų on the specimen, and the exposure time was 1.5 s. Each image was fractionated into 23 frames.

Cryo-EM data optimization and screening was performed on a Talos (200 kV using a Falcon 2 direct electron detector). Cryo-EM data collection was performed on a Titan Krios electron microscope (FEI) operated at an acceleration voltage of 300 kV using a Falcon 3 direct electron detector camera (FEI). Images were recorded with a pixel size of 1.08 Å, 1 s exposures at 93 electrons/Ų/sec dose rate, 39 fractions. A total of 534 images were used for analysis and processing. Motioncorr 2 was used for motion correction^{54 (link)} and GCTF was used for CTF estimation as implemented in RELION 3. FLAG-c-FLIP_S:FADD:Caspase-8 complexes were initially picked using CrYOLO²⁶ , yielding a dataset of 12918 particles. Reference-free 2D Class averages were generated from these data in RELION 3 and 10 good classes then used as templates for auto-picking of particles in RELION 3, yielding a dataset of 46023 particles (Supplementary Table 1). The single-particle analysis was performed in RELION 3^{27 (link)}, as outlined in the flowchart in Supplementary Fig. 17, resulting in a final post-processed EM map with a final resolution of ~13 Å.