Vitrobot plunge freezer

Cryo-EM grids of KimA in SMALP at 1.1 mg/mL with 50 μM cyclic di-AMP were prepared in a FEI Vitrobot plunge freezer at 10 °C and 90% humidity, using Quantifoil R2/2 holey carbon grids (Quantifoil Micro Tools), pre-treated in chloroform for 1 to 2 h and freshly glow-discharged. The grid was blotted for 9 sec and plunge-frozen in liquid ethane. Images were collected automatically using EPU (Thermo Scientific), on a FEI Titan Krios operating at 300 kV and aligned as previously described^{40 (link)}, with a Gatan K2 camera in counting mode and with an energy filter. The nominal magnification of 130,000x yielded a pixel size at the specimen of 1.077 Å. Each micrograph was recorded as a movie stack with 40 frames over 8 sec, with a calibrated dose of ~1.77 e^–/Ų per frame and defocus values between −0.5 and −3.2 μm.

The purification of the Frh complex from Methanothermobacter marburgensis and the preparation of the grids for the electron microscope data collection were performed as described (Mills et al., 2013 (link)). Briefly, 3 μl of a 0.7 mg/ml Frh sample in the presence of 10 mM F₄₂₀ was applied to freshly glow discharged Quantifoil R1/4 holey carbon grids (Quantifoil Micro Tools, Jena, Germany). The grids were blotted in an FEI Vitrobot plunge-freezer. Data was collected on an FEI Tecnai Polara operating at 300 kV, using a back-thinned FEI Falcon II direct electron detector. The microscope was carefully aligned as previously described (Mills et al., 2013 (link)) and the Falcon II camera was calibrated at the desired nominal magnification of 78,000×. The calibrated magnification on the 14 µm pixel camera was 106,000, resulting in a 1.32 Å pixel size at the specimen. The camera system was set up to record 18 frames/sec as previously described (Bai et al., 2013 (link)). Videos were collected for 1.5 s with a total of 24 frames with a calibrated dose of 3.5 e⁻/Ų per frame, at various defocus values in the range between 0.8 and 3.8 μm.