Cryo-EM Sample Preparation and Imaging

Quantifoil R2/1 or R1.2/1.3 300 mesh holey carbon grids were glow discharged for 2 minutes at 20 mA. The freshly prepared 3 µL protein samples at a concentration of 0.5 mg/ml were added to the carbon grids and incubated for 10 seconds before plunging into liquid ethane using FEI Vitrobot Mark IV plunger at 20°C with 100% humidity. The grids were blotted for 3-5 seconds at zero blot force. Cryo-EM data acquisition of the frozen sample was carried out using a Thermo Scientific™ Talos Arctica transmission electron microscope at 200 kV at a nominal magnification of 42,200 x, equipped with a K2 Summit Direct Electron Detector. LatitudeS automatic data collection software (Gatan Inc) was utilized to collect the final 746 digital micrographs at a pixel size of 1.2 Å with a total electron dose of about 40 e-/Ų at the defocus range of −1.25 and −3.5 µm, under a calibrated dose of about 2 e-/Ų per frame. Movies were recorded for 8 secs with 20 frames. All the collected micrographs were then motion corrected using “dosefgpu_driftcorr” using MotionCor2 software (39 (link)).