Aliquots of NC22/17-HA 131E ectodomain (4 uL, 0.18 mg/mL, in 20 mM Tris pH 8.0, 150 mM NaCl.) were applied to graphene oxide (GO) grids (GO on Quantifoils R1.2/1.3 400 mesh copper grids, R1.2/1.3). In the Vitrobot Mark IV (Thermo Fisher Scientific), the humidity was set at 100%, the protein solution was applied to the grid and there was a wait of 1 s before blotting. A blot force of 0 and blot time of 2 s were applied to blot the grid after waiting. After blotting, the grid was plunged into precooled liquid ethane at a liquid nitrogen temperature (69 (link)).
Images for complex were recorded using FEI Titan Krios microscope (Thermo Fisher Scientific) operating at 300 kV with a Gatan K3 Summit direct electron detector (Gatan Inc.) at Shanxi Academy of Advanced Research and Innovation. The automated software (EPU)3 was used to collect 5,746 movies for complex in super-resolution mode at a nominal magnification of 105,000× with a calibrated pixel size of 0.85 Å and at a defocus range between −1.0 and −2.0 um. Each movie has a total accumulate exposure of 60 e-/Å2 fractionated in 32 frames of 1.38 s exposure (70 (link)).
Images for complex were recorded using FEI Titan Krios microscope (Thermo Fisher Scientific) operating at 300 kV with a Gatan K3 Summit direct electron detector (Gatan Inc.) at Shanxi Academy of Advanced Research and Innovation. The automated software (EPU)3 was used to collect 5,746 movies for complex in super-resolution mode at a nominal magnification of 105,000× with a calibrated pixel size of 0.85 Å and at a defocus range between −1.0 and −2.0 um. Each movie has a total accumulate exposure of 60 e-/Å2 fractionated in 32 frames of 1.38 s exposure (70 (link)).
