Tecnai tf20 electron microscope

Holey gold film (Russo and Passmore, 2014 (link)) coated EM grids with 2 µm holes were prepared following a previously described procedure (Marr et al., 2014 (link)). The S-protein solution contained 0.4 to 0.8 mg/mL protein in 10 mM Tris, pH 8.0 and 150 mM NaCl. Vitrification of the cryo-EM specimen was performed using a Gatan Cryoplunge 3 device. For each cryo-EM specimen, 3 µL of S-protein solution was applied to a glow-discharged (2 min, 15 mA) holey grid. The grids were blotted for 13 s at 100% humidity and 295 K and then plunge-frozen in a 50:50 ethane:propane mixture (Tivol et al., 2008 (link)) at 77 K. Specimen screening and optimization was done with an FEI Tecnai TF20 electron microscope equipped with a Gatan K2 summit direct detector. The final high-resolution data collection was performed with a Thermo Fisher Scientific Titan Krios G3 300 kV microscope equipped with a Falcon 3EC direct detector. The final high-resolution data were collected at 75000x nominal magnification, resulting in a calibrated pixel size of 1.06 Å. Each movie was recorded in counting mode with a 60 s exposure and saved in 30 fractions. The exposure rate was adjusted to 0.8 electrons per pixel per second, resulting in a total exposure of 42.7 electrons per Ų. The data were collected with a 1.8 to 2.2 µm defocus. A total of ~4000 movies were collected for the final high-resolution dataset.

Ten millilitres of each filtered phage suspension were centrifuged at 40,000 x g for 90 min at 4 °C. The supernatants were carefully removed and the pellets re-suspended gently in 0.5 ml of 0.1 M ammonium acetate (pH 7.3). The suspensions were stored overnight at 4 °C for negative staining and vizualisation by TEM. Three hundred-mesh carbon-coated nickel grids were dropped onto 50–100 μl of phage suspension. The grids were allowed to adsorb phage suspension for 1–2 min and were washed three times with dH₂O for 10 s. Excess fluid was removed using Whatman filter paper and the grids were negatively stained with 2% ammonium molybdate for 30 s. Excess staining solution was removed with Whatman filter paper and the grids allowed to air-dry at room temperature for 15 to 20 min. The samples were examined by TEM using a FEI Tecnai TF20 electron microscope at 200 kV and Gatan Microscopy Suite Software.