Bioquantum k3 imaging filter

Purified substrate-bound Bam complex as described above was applied to glow-discharged Quantifoil R 2/1 holey carbon 400-mesh copper grids (Quantifoil). Grids were blotted for 4–5 s at 100% humidity with the blot force set to 16, and flash frozen by liquid nitrogen-cooled liquid ethane using a Thermo Fisher Scientific Vitrobot Mark IV (Thermo Fisher Scientific). The grid was then loaded onto a Titan Krios G3i electron cryo-microscope (Thermo Fisher) operated at 300 kV accelerating voltage. Image stacks (movies) were recorded on a Gatan Bioquantum K3 Imaging Filter (Gatan, USA) using the super-resolution counting mode and the calibrated magnification of 58717× using SerialEM⁴⁹ . The slit of the energy filter was set to 25 eV, with a defocus range between 1.1 and 2.8 μm. The electron dose rate was 17 e−/physical pixel per second, and the subframe time was set to 0.06 second. A total exposure time of 3 second resulted in 50 subframes per image stack. The total electron dose was 70 e−/Ų (~1.4 e−/Ų per subframe). The multi-shot scheme in SerialEM was used for data collection, with settings of 4 holes per stage move, and 5 shots per hole, which greatly sped up the data collection. The data collection for both structures (4.1 Å and 6.5 Å) was performed in the same manner.

Before grid preparation, 1 mM ATPγS and 2.5 mM MgCl₂ were added to the protein sample with incubation on ice for 1 h. A 3.5 μl drop containing the NLRP3–NEK7–ASC complex was loaded onto a glow-discharged Quantifoil grid (R1.2/1.3 400-mesh gold-supported holey carbon, Electron Microscopy Sciences), blotted for 3–4 s under 100% humidity at 4 °C and plunged into liquid ethane with Mark IV Vitrobot (ThermoFisher). Automated data collection was performed using SerialEM (Mastronarde, 2005), and videos were acquired on a Titan Krios microscope (ThermoFisher) operating at 300 keV and equipped with a BioQuantum K3 Imaging Filter (Gatan, slit width 20 eV). Videos were recorded with a K3 Summit direct electron detector (Gatan) operating in super-resolution mode at ×81,000 magnification (0.53 Å per pixel). All videos were exposed at a total dose of 50.4 e Å⁻² over 40 frames with defocus range between −1.0 and −2.0 μm.