K2 direct electron detector camera

For cryo-EM experiments, LACV-L FL in complex with pre-annealed 3′ (1–16) and 5′ (9–16) vRNA at 0.2 mg/ml was mixed with 5′ (1–10) vRNA hook in a 1:2 molar ratio. UltraAuFoil grids 300 mesh, R 1.2/1.3 were negatively glow-discharged at 30 mA for 1 min. 3.5 µl of the sample was applied on the grids and excess solution was blotted away with a Vitrobot Mark IV (FEI) (blot time: 2 s, blot force: 1, 100% humidity, 20 °C), before plunge-freezing in liquid ethane. Grid screening and cryo-EM initial datasets were collected on a 200 kV Thermofischer Glacios microscope equipped with a Falcon II direct electron detector.
A high-quality cryo-EM grid pre-screened on a 200 kV Thermofischer Glacios microscope was used to collect data on a Thermofischer Titan Krios G3 operated at 300 kV equipped with a Gatan Bioquantum LS/967 energy filter (slit width of 20 eV) coupled to a Gatan K2 direct electron detector camera^{31 (link)}. Automated data collection was performed with SerialEM using a beam-tilt data collection scheme^{32 (link)}, acquiring one image per hole from nine holes before moving the stage. Micrographs were recorded in super-resolution mode at a 165,000× magnification giving a pixel size of 0.4135 Å with defocus ranging from −0.8 to −3.5 µm. In total, 16,498 movies with 40 frames per movie were collected with a total exposure of 50 e⁻/Å^{2 (link)} (Supplementary Table 2).

Cryo-grids were loaded into a Talos Arctica TEM (Thermo Fisher Scientific) operating at 200 kV, equipped with the K2 direct electron detector camera (Gatan) and sample autoloader. Total exposure was split into 250 ms frames with a total cumulative dose of ~50 e^-/Ų. Exposure magnification of 36,000 was set with the resulting pixel size of 1.15 Å at the specimen plane. For BG505-SOSIP-T33_dn10 nanoparticle imaging the nominal defocus range was -0.6 to -2.0 μm. The range was -0.8 to -2.0 for BG505-SOSIP-I53_dn5. Automated data collection was performed using Leginon software [85 (link)]. The data collection details for the acquired datasets are presented in S3 Table.