ACIII Cryo-EM Sample Preparation Protocol

An aliquot of 3 μl of an ACIII sample at 1 mg ml⁻¹ was applied to freshly glow discharged C-Flat multihole holey carbon grids (Electron Microscopy Sciences). Grids were blotted for 9 s at 90% humidity and 10 °C in an FEI Vitrobot plunge freezer. Cryo-EM images were collected on a FEI Titan Krios operating at 300 kV aligned as described^{42 (link)}. The microscope was equipped with a Gatan K2 Summit electron detector and an energy filter. Images were recorded manually in counting mode at a nominal magnification of ×135,000, yielding a pixel size of 1.035 Å at the specimen. Movies were collected for 8 s with a total of 40 frames and a calibrated dose of about 1.8 e⁻ Å⁻² per frame (total dose 72 e⁻ Å⁻²), at defocus values between −0.6 and −4.0 μm.