Cryo-EM Imaging of Amyloid Fibrils

Cryo-EM grids were prepared using a Vitrobot Mark IV (ThermoFisher Scientific) with 95% humidity at 4°C. Amyloid fibrils (3 µL aliquots) were applied onto glow-discharged, 300 mesh, copper Quantifoil grids. After blotting, grids were plunge frozen in liquid ethane cooled by liquid nitrogen. Samples were imaged on a Titan Krios (ThermoFisher Scientific) transmission electron microscope, operated at 300 kV and equipped with a Gatan Quantum-LS imaging energy filter (GIF, 20 eV energy loss window; Gatan Inc.). Images were acquired on a K2 Summit electron counting direct detection camera (Gatan Inc.) in dose fractionation mode (50 frames) using the Serial EM software (Mastronarde, 2005 (link)) at a magnification of 165,000× (physical pixel size 0.831 Å) and a total dose of ~69 electrons per square angstrom (e^-/Ų) for each micrograph. Micrographs were drift-corrected and dose-weighted using MotionCor2 (Zheng et al., 2017 (link)) through the Focus interface (Biyani et al., 2017 (link)). Additional data collection parameters are detailed in Table 1.