Cryo-EM complex and grid preparation

1.47 mg of RSV preF with 1.45 mg of MxR Fab were mixed and incubated overnight at 4 °C with gentle rocking, before SEC purification over a 10/300 Superose 6 column (Cytiva, cat#29091596). A very broad peak eluted, with the nine largest fractions concentrated to 0.22 mg/mL using a 10 kDa cutoff Amicon ultrafiltration unit (Sigma-Aldrich, cat#UFC8030). For 3 × 1, we incubated 50 µg of HPIV3 preF with 150 µg of 3 × 1 Fab overnight at 4 °C with gentle rocking before SEC purification over a Superdex 200 16/600 SEC column (Cytiva, cat#28989335). A single narrow high molecular weight peak was eluted and was concentrated to 0.4 mg/mL using a 10 kDa cutoff Amicon ultrafiltration unit.

Both complexes were frozen on Quantifoil 1.2/1.3 UltrAu 300 mesh grids (Electron Microscopy Sciences, cat#Q350AR13A) using a Vitrobot Mk. IV (Thermo Fisher) at 22 °C and 100% humidity. Dodecyl-β-d-maltoside (DDM) was added to the sample (0.05% final concentration) 30 min prior to the start of freezing, and a PELCO easiGlow™ (Tedpella, Cat#91000) was used to glow discharge the grids. The final MxR:RSV grids used for the collection were frozen with 4 µL of the sample at 0.21 mg/mL, a 14 s blot time, 0 blot force, and a 5 s wait between application and blotting. The final 3 × 1:HPIV3 grids used for the collection were frozen with 4 µL of the sample at 0.19 mg/mL, a 12 s blot time, 0 blot force, and a 15 s wait between application and blotting.