Cryo-EM sample preparation and data acquisition

Freshly prepared OpuA nanodiscs were concentrated to at least 1 mg/ml using a Vivaspin 500 (50 kDa cutoff) concentrator. Before freezing, OpuA was diluted to 1 mg/ml and respective additives were added (e.g., 100 μM glycine betaine, 10 μM cyclic-di-AMP, 10 μM MgAMP-PNP, or 10 mM MgATP). Holey carbon grids (Quantifoil; Au R1.2/1.3, 300 mesh) were glow-discharged for 20 s at 5 mA. Sample (2.8 μl) was applied on the grids, after which they were blotted for 3 to 5 s in a Vitrobot Mark IV (Thermo Fisher Scientific) at 15° to 22°C and 100% humidity and plunge-frozen into a liquid ethane/propane mixture. The grids were then stored in liquid nitrogen until further use.

Data collection was performed in-house on a 200-keV Talos Arctica microscope (Thermo Fisher Scientific) equipped with a K2 and a post-column BioQuantum energy filter (Gatan) operated in zero-loss mode, with a 20-eV slit, and a 100-μm objective aperture. Automatic collection was done at a calibrated magnification of 49,407× (1.012 Å pixel size) and a nominal defocus range from −0.8 to −1.9 μm using EPU (Thermo Fisher Scientific). Holes were selected with the help of an in-house–written script that calculates ice thickness within DigitalMicrograph (Gatan). Each movie consisted of 60 frames with a total exposure time of 9 s and a dose of 53 electrons/Ų (0.883 e⁻/Ų per frame). FOCUS software (38) was used for on-the-fly quality control, and settings were adjusted if necessary.