micrographs - multiframe

TIFF

1994

(None, None)

SIGNED BYTE

(1.06, 1.06)

Cryo-EM data collection of NLRP1 CT filaments was conducted at the HMS Cryo-EM Center. Purified NLRP1 CT filaments (A280 = 0.50; 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM TCEP) were loaded onto a glow-discharged C-flat grid (CF-1.2/1.3 400-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. Movies were acquired using a Titan Krios microscope (ThermoFisher) at an acceleration voltage of 300 keV equipped with a BioQuantum K3 Imaging Filter (slit width 25 eV), and a K3 direct electron detector (Gatan) operating in counting mode at 81,000 x (1.06 Å pixel size). Automated data collection with SerialEM varied the defocus range between −0.8 to −2.2 μm with four holes collected per stage movement through image shift. All movies were exposed with a total dose of 52.3 e-/Å2 for 3.5 s fractionated over 50 frames. Gain reference file is "gain.mrc".

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