UltrAuFoil R1.2/1.3 300-mesh grids were glow discharged at 40 mA for 5 min using a GloQube glow discharge system (Quorum) and coated with graphene oxide flakes (Sigma). Next, 4 μl of 40 ng μl–1 DDK was applied to each grid for 30 s before double-sided blotting for 4 s and plunge-freezing in liquid ethane using a Vitrobot Mark IV (FEI) operated at room temperature with 90% humidity. A total of 9,469 videos were collected at ×165,000 magnification (yielding a pixel size of 0.84 Å at specimen level) on a Titan Krios electron microscope equipped with a K2 Summit direct electron detector (Gatan Inc.).
A total of 2,967,226 particles were picked from motion-corrected video sums using crYOLO65 (link). CTF was estimated using Gctf64 (link), and 2D classification was performed after extraction of particles in a 240-pixel box in RELION-3.0.7 (ref. 39 ).
A total of 2,967,226 particles were picked from motion-corrected video sums using crYOLO65 (link). CTF was estimated using Gctf64 (link), and 2D classification was performed after extraction of particles in a 240-pixel box in RELION-3.0.7 (ref. 39 ).
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