Titan krios g2 tem

For each grid, 3 μL of purified protein solution of CTSL-treated or untreated SARS-CoV-2 S proteins were applied to newly glow-discharged holy carbon film grids (R1.2/1.3, 200 meshes, Au, Quantifoil, Germany) or holy Ni-Ti film grids^{48 (link)} (R1.2/1.3, 300 meshes, Au, Zhenjiang Lehua Electronic Technology Co., LTD, China). Then the grid was blotted and vitrified by plunge freezing into liquid ethane using Vitrobot Mark IV (Thermo Fisher Scientific, USA) at 4 °C and 100% humidity. All movies were collected on a Titan Krios G2 TEM (Thermo Fisher Scientific, USA) operated at 300 KV on EF-TEM mode with nominal magnification set to be ×105,000, resulting in a calibrated physical pixel size of 1.36 Å on a specimen level. The total dose was set to be 60 e−/Ų, with an exposure time of 15 s. The movies were acquired by Gatan K2 Summit DDD detector (Gatan Company, USA) equipped with a GIF Quantum energy filter, with a silt width of 20 eV, operated in super-resolution mode, resulting in a pixel size of 0.68 Å for output super-resolution movies. SerialEM^{49 (link)} with in-house scripts were used for data collection^{50 (link)}, defocus values for either samples were set between −1.0 and −2.0 μm.

Cryo-ET samples of single- and double-layered Octa and protein/Octa lattices were prepared on lacey carbon film grids (Cu-200LC, Electron Microscopy Sciences, and Cu-200LN, Pacific Grid-Tech) using the Leica EM GP plunge freezer (Leica, Inc.) at 90% humidity and 4 °C. In brief, lattice solutions (4 μL) were incubated with glow-discharged grids and rapidly plunged into liquid ethane after blotting. Samples were screened by Zeiss Libra 120 Plus TEM (Carl Zeiss SMT GmbH) with in-column energy filter and a 4 k × 4 k Gatan UltraScan 4000 CCD camera, and operating at 120 kV and a low-dose condition. High-resolution tilt series were acquired by a Titan Krios G2 TEM (ThermoFisher Scientific) with a Gatan energy filter (Gatan, Inc.), operating at 300 kV. Micrographs were recorded on a Gatan K3 direct electron detector operated in super-resolution mode at a nominal magnification of 53 K (1.46 Å/pixel) with defocus ~3 μm using SerialEM^{72 (link)}. Non-tilt micrographs were acquired with 3.0 s exposure time, 0.15 s each frame, at a dose rate of ~8 e⁻/Ų/s. Tilt series were collected from −51° to +51° at a 3° increment, starting at +21°, with 1.0 s exposure time, 0.15 s each frame, at a dose rate of ~8 e⁻/Ų/s.