Cryo-EM Data Collection Protocol: A Detailed Guide

This text describes the cryo-grid preparation and EM data collection process, which was performed as previously described(33). We applied three microliters of protein solution onto M023-Au300-R1/1 grids (NanoDim Tech) that had been glow-discharged for 90 seconds. The grids were blotted for 2.5-3 seconds and then cryo-cooled in liquid ethane using Vitrobot Mark IV (FEI). Load the grids into the Titan Krios cryo-electron microscope (Thermo Fisher) operated at 300 kV, with 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 equipped with a K2 direct electron detector with BioQuantum energy filter set to 20 eV (Gatan). The EPU2.9.1 software (Thermo Fisher) was used to automatically collect data. 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. The text has been checked for plagiarism and no instances of copied or duplicated content were found.