Titan krios g4 electron microscope

Cryo-EM was performed at the Ian Holmes Imaging Centre, Bio21 Institute of Molecular Science and Biotechnology, The University of Melbourne. Prior to EM specimen grid preparation, the pre-fusion stabilised S trimer (Hexapro) was incubated with the BP10 nanobody construct at a 6:1 molar ratio for 30 min at room temperature. The complex was purified over SEC and concentrated to 3 mg/mL. Grids (Quantifoil Cu R1.2/1.3, 300 mesh) were glow discharged in air at 15 mA for 60 s using an EasiGlow glow discharge apparatus (Pelco, USA). The 4 μL samples were applied to the grids at 4°C and 100% humidity, and then plunge frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). Data was collected on a Titan Krios G4 electron microscope (Thermo Fisher Scientific) operated at 300 kV and equipped with a Falcon 4 detector (Thermo Fisher Scientific) and Bioquantum energy filter (Gatan, Pleasanton, USA). The dataset was collected at a pixel size of 0.66 Å with 13,806 movies containing 40 frames each with a total exposure of 50.0 e/Ų. The acquisition was performed at 155Kx indicated magnification using an energy filter slit width of 10 eV to remove contributions from inelastically scattered electrons. All data were collected using EPU software Version 2.9 (Thermo Fisher Scientific) using a 9-hole beam-image shift acquisition scheme with one exposure per hole.