R2 1 au g200f4 grids

The LASV L-Cstrep protein with a concentration of 3 µM in assay buffer (100 mM HEPES(NaOH) pH 7.0, 50 mM NaCl, 50 mM KCl, 2 mM MnCl₂ and 2 mM dithiothreitol) was mixed sequentially with single-stranded 5′ (0–19) vRNA and single-stranded 3′ (1–19) vRNA in 1.7-fold and primer C8 in 3.3-fold molar excess (all RNAs are listed in Supplementary Table 1). After 45 min incubation on ice, the reaction was started by addition of NTPs (0.25 mM GTP/ATP/UMPNPP and 0.125 mM CTP). After incubation at 30 °C for 2 h, 3 µL of the reaction was applied to glow-discharged Quantifoil R2/1 Au G200F4 grids, immediately blotted for 2 s using an FEI Vitrobot Mk IV (4 °C, 100% humidity, blotting force–10) and plunge frozen in liquid ethane/propane cooled to liquid nitrogen temperature.

The LASV L-Cstrep protein with a concentration of 1.4 µM in assay buffer (100 mM HEPES(NaOH) pH 7.0, 50 mM NaCl, 50 mM KCl, 2 mM MnCl₂ and 2 mM dithiothreitol) was mixed sequentially with single-stranded 5′ (0–19) vRNA and single-stranded 3′ (1–19) vRNA in 1.2-fold and primer St1 in 7.1-fold molar excess (all RNAs are listed in Supplementary Table 1). After 45 min incubation on ice, the reaction was started by the addition of NTPs (0.25 mM GTP/ATP). After incubation at 30 °C for 2 h, 3 µL of the reaction was applied to glow-discharged Quantifoil R2/1 Au G200F4 grids, immediately blotted for 2 s using an FEI Vitrobot Mk IV (4 °C, 100% humidity, blotting force–10) and plunge frozen in liquid ethane/propane cooled to liquid nitrogen temperature.