Cryo-EM Sample Preparation and Imaging

EM grids (Lacey
Carbon Film, Electron Microscopy Sciences #LC200-Cu) were glow-discharged
(Emitech K350 unit at 20 mA for 30 s), deposited with 4 μL of
lipid dispersion, blotted, and then plunged into liquid ethane using
a Vitrobot (Mark IV, Thermo Fisher Scientific). Images were collected
on a Titan Krios G3 (Thermo Fisher Scientific) operated at 300 keV
equipped with a K3 detector and a 1067HD BioContinuum energy filter
(Gatan) with a 15 eV slit width. Dose-fractionated images were acquired
using SerialEM^{43 (link)} in low dose counting mode
with a total dose of 50 e/Ų at 2.16 Å/pixel
and 4 μm nominal defocus. Images were motion- and CTF-corrected
in Warp.^{44 (link)} Representative images were prepared
in ImageJ.^{45 (link)}