Cryo-EM Imaging of KimA in SMALP

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.