Fei talos transmission electron microscope

BINTOXB/9+proCDTb at 0.03 mg/mL or CDTb double heptamer (from reaction with no Fab or BINTOXB/19 Fab) at 0.05 mg/mL was applied to CF400-Cu grids (Electron Microscopy Sciences) that were plasma-cleaned for 30 s in a Gatan Solarus 950 with a 4:1 O₂:H₂ ratio and allowed to bind to the support for 30 s. The grid was then washed twice with 20 mM Tris (pH 8), 150 mM NaCl, 6 mM CaCl₂ and then stained with NanoW (Nanoprobes). Grids were imaged in an FEI Talos transmission electron microscope (Thermo Fisher Scientific) using a Ceta16M detector. Micrographs were collected at a magnification of ×92,000, corresponding to a pixel size of 1.63 Å/pixel. Contrast transfer function estimation, particle picking, 2D classification, ab initio reconstruction, heterogeneous refinement, and homogeneous refinement were performed in CryoSPARC v3.3.1 (46 (link)).

PEDV CV777 S at a concentration of 0.4 mg/ml was incubated with 2 mM 3′-sialyllactose (Sigma-Aldrich) in 2 mM Tris (pH 8.0), 200 mM NaCl, 0.02% NaN₃, 0.01% Amphipol A8-35 for 3 h at 4 °C. A 3-μl volume of the spike and 3′-sialyllactose mixture was deposited on a plasma-cleaned CF-2/2 grid before being blotted for 6 s and plunge-frozen in liquid ethane using a Vitrobot Mark IV instrument. The frozen grid was imaged using an FEI Talos transmission electron microscope (Thermo Scientific) equipped with a Ceta 16M detector. Micrographs were collected manually using TIA v4.14 (Thermo Scientific) at a nominal magnification of ×92,000, corresponding to a calibrated pixel size of 1.63 Å/pixel. A full description of the EM data collection parameters can be found in Table 1. CTF estimation, particle picking, 2D classification, ab initio 3D reconstruction, and 3D refinement were performed in cisTEM (49 (link)).