Cryo-EM Sample Preparation for SIDT2 Proteins

Holey carbon grids (Quantifoil Au 300 mesh, R1.2/1.3) were glow-discharged in the Plasma Cleaner PDC-32G-2 (Harrick Plasma Company) with a vacuum for 2 min and mid force for 30 s. Aliquots (4 µl) of SIDT2 proteins were placed on the glow-discharged grids, which were then blotted for 3 s and flash frozen in liquid ethane cooled by liquid nitrogen using Vitrobot Mark IV (Thermo Fisher Scientific) at 8 °C and 100% humidity. The grids were loaded onto a 300 kV Titan Krios (Thermo Fisher Scientific Inc.) equipped with K3 Summit detector (Gatan) and GIF Quantum energy filter. Images were automatically collected using AutoEMation^{49 (link)} in super-resolution mode at a nominal magnification of 81,000 × (64,000 × for apoSIDT2-pH 7.4 dataset), with a slit width of 20 eV on the energy filter. A defocus series ranging from −1.3 μm to −1.8 μm was used. Each stack was exposed for 2.56 s with an exposure time of 0.08 s per frame, resulting in a total of 32 frames per stack and the total dose was approximately 50 e-/Ų for each stack. The stacks were motion corrected with MotionCor2^{50 (link)} and binned 2 fold, resulting in a pixel size of 1.0825 Å/pixel (1.0979 Å for apoSIDT2-pH 7.4 dataset). Meanwhile, dose weighting was performed^{51 (link)}. The defocus values were estimated with Gctf^{52 (link)}.