Cryo-EM Protein Sample Preparation

For cryo-EM sample preparation, three microliters of each protein were placed on glow-discharged holey carbon grids (Quantifoil Au R2.1/3.1, 300 mesh). The grid was blotted with filter paper for 3.5 s in a chamber set with 100% humidity at 8 °C to remove the excess sample and then plunge-frozen in liquid ethane cooled by liquid nitrogen with the Vitrobot Mark IV system (ThermoFisher Scientific). Cryo-EM specimens were imaged on a 300-kV Titan Krios electron microscope (ThermoFisher Scientific) using a normal magnification of 81,000 rpm. Movies were recorded using a Gatan K3 detector equipped with a GIF Quantum energy filter (slit width 20 eV) at the super-resolution mode, with a physical pixel size of 1.087 Å. Each stack of 32 frames was exposed for 2.56 s, with a dose rate of ∼23 counts/second/physical-pixel (∼19.5 e-/second/Ų) for each frame using EPU (ThermoFisher Scientific). All 32 frames in each stack were aligned and summed using the whole-image motion correction program MotionCor2^{60 (link)} and binned to a pixel size of 1.087 Å. The defocus value for each image varied from −1.5 to −2.0 μm and was determined by Gctf^{61 (link)}.