Cryo-EM sample preparation and imaging

Ctf19c samples were applied to glow-discharged C-flat grids (CF-1.2/1.3–3C; Electron Microscopy Sciences). In all cases, 3.5 μL of protein solution were applied, and grids were blotted from both sides for 4 s before vitrification in liquid ethane using a Cryoplunge 3 instrument (Gatan) operating at 80–90% humidity. For screening of sample preparations and generation of initial maps, we used a Tecnai F20 (FEI) microscope operating at 200 kV. Images, collected using the UCSF Image4 software package (Li et al., 2015), were recorded on a K2 Summit electron detector (Gatan) operating in super-resolution movie mode (50 frames, 0.2 s/frame,~60 electrons per Ų total dose, 0.64 Å/super-resolution pixel).

For collection of high-resolution data, we used an FEI Polara microscope (FEI) operating at 300 kV. Images, collected using the SerialEM software package (Mastronarde, 2005), were recorded on a K2 Summit electron detector operating in super-resolution movie mode (40 frames, 0.2 s/frame, 52 electrons per Ų total dose, 0.615 Å/super-resolution pixel). In total, we collected 15,439 movies over three sessions.