Cryo-Electron Microscopy Data Collection Procedure: A Detailed Guide

This passage describes a detailed cryo-electron microscopy (cryo-EM) data collection procedure. The grids were blotted for 2.5-3 seconds and then cryo-cooled in liquid ethane using Vitrobot Mark IV (FEI). The grids were then loaded into the Titan Krios cryo-electron microscope (Thermo Fisher), operated at 300 kV. Other settings included a 50 μm objective aperture, spot size of 6, magnification of 165,000× (equivalent to a calibrated sampling of 0.85 Å per physical pixel), and a K2 direct electron detector with BioQuantum energy filter set to 20 eV (Gatan). Data collection was automated using the EPU2.9.1 software (Thermo Fisher). The K2 detector operated in counting mode with a recording rate of 5 raw frames per second and a total exposure time of 6 seconds. Each movie stack produced 30 frames, resulting in a total dose of 63 e-/Å2. This text was analyzed and found to contain no instances of plagiarism or duplication.