Cryo-EM Sample Preparation for Proteins

Proteins for cryo-EM were concentrated to ∼3 mg/ml. Three microliters of the protein solution were applied on a glow-discharged holey carbon grids (200 mesh Quantifoil R2/2 molybdenum), which had been treated in a DII-29020HD (JEOL) for 40 s, and then plunged into liquid ethane at –178 °C using an EM GP2 plunger (Leica, Microsystems). The applied parameters were blotting time 6s and 90% humidity at 4 °C. The data were collected on a CRYO-ARM300 (JEOL) electron microscope at 300 kV equipped with a K3 camera (Gatan). An in-column energy filter with a slit width of 20 eV was inserted for acquisition of movie frames. The movies were recorded using JADAS software (JEOL) (37 (link)) at a nominal magnification of 60K in counting mode and a pixel size of 0.814 Å at the specimen level with a dose rate of 17.7 e-per physical pixel per second, corresponding to 26.7 e-per Ų per second at the specimen level. The exposure time was 1.5 s, resulting in an accumulated dose of 40.0 e-per Ų. Each movie includes 20 fractioned frames.