micrographs - multiframe

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1208

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Cryo-EM data collection of CARD8 CT filaments was conducted at the Pacific Northwest Center for Cryo-EM (PNCC). Purified CARD8 CT filaments (A280 = 0.75; 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM TCEP) were loaded onto a glow-discharged Quantifoil grid (R1.2/1.3 400-mesh copper-supported holey carbon, Electron Microscopy Sciences), blotted for 4–5 s under 100% humidity at 4 °C, and plunged into liquid ethane using a Mark IV Vitrobot (ThermoFisher). Grids were screened for ice and particle quality prior to data collection. 1,208 movies were acquired using a Titan Krios microscope (ThermoFisher) at an acceleration voltage of 300 keV equipped with a Falcon 3EC direct electron detector (ThermoFisher) operating in counting mode at 96,000 x (0.8315 Å pixel size). Automated data collection with EPU varied the defocus range between −0.8 to −2.2 μm with three movies collected per hole through image shift prior to stage movement. All movies had an exposure time of approximately 40 s for a total dose of 40 e-/Å2 fractionated over 50 frames. Multi-frame movies are saved gain-corrected. Readme.docx provided.

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