Cf400 cu grid

Purified SARS-CoV-2 spike was diluted to a concentration of 0.04 mg/mL using 2 mM Tris pH 8.0, 200 mM NaCl and 0.02% NaN₃ before being applied to a plasma cleaned CF400-Cu grid (Electron Microscopy Sciences). Protein was then stained using methylamine tungstate (Nanoprobes) before being allowed to dry at room temperature for 15 minutes. This grid was imaged in a Talos TEM (Thermo Fisher Scientific) equipped with a Ceta 16M detector. Micrographs were collected using TIA v4.14 software at a nominal magnification of 92,000×, corresponding to a calibrated pixel size of 1.63 Å/pix. CTF estimation, particle picking and 2D class averaging were performed using cisTEM³⁹ .

The postfusion gB was deglycosylated by digestion with Endo H (10% w/w) for 12 hours at 4°C and purified using a Superose6 10/300 column. Deglycosylated gB was mixed with a molar excess of 3–25 Fab and run over a Superose6 10/300 column (GE Healthcare) using 2 mM Tris pH 8.0, 200 mM NaCl, 0.02% NaN₃. A CF400-Cu grid (Electron Microscopy Sciences) was plasma cleaned for 30 seconds in a Solarus 950 (Gatan) using a 4:1 mixture of O₂ to H₂. The purified complex was diluted to a concentration of 0.025 mg/mL and mixed with additional 3–25 Fab to fully saturate the gB trimer before being deposited onto the prepared grid. Micrographs were collected using a 200 kV Talos transmission electron microscope (Thermo Fisher) equipped with a Ceta 16M detector (Thermo Fisher) at a magnification of 92,000×, corresponding to a calibrated pixel size of 1.63 Å. Data were collected manually using TIA v4.14 (Thermo Fisher) at a defocus range of 1.8 to 3.1 μm. CTF-estimation, particle picking and preliminary 2D classification were performed using cisTEM [63 (link)] before particles were exported into cryoSPARC v2.9.0 [64 (link)] for ab initio model generation and 3D classification.

Postfusion hMPV F was heat-treated at 70 °C for 10 min, then applied to a CF-400-Cu grid (Electron Microscopy Sciences) that had been plasma cleaned for 45 s in a Solarus 950 plasma cleaner (Gatan) with a 4:1 ratio of O₂/H₂. The grid was stained using methylamine tungstate (Nanoprobes). Prefusion-stabilized hMPV F DS-CavEs2 was incubated with a two-fold molar excess of MPE8 Fab in 1× PBS at room temperature for 30 min. The hMPV-F:Fab complexes were diluted to a concentration of 0.03 mg/mL in 2 mM Tris pH 8.0, 200 mM NaCl and 0.02% NaN₃, then deposited on a CF-400-Cu grid. Grids were imaged at a magnification of 92,000× (corresponding to a calibrated pixel size of 1.63 Å/pix) in a Talos F200C TEM microscope equipped with a Ceta 16 M detector (Thermo Fisher Scientific). CTF-estimation and particle picking were performed in cisTEM³⁵ . Particles were then exported to cryoSPARC v2.15.0 for 2D classification^{36 (link).}