Cryo-EM Imaging Using Leica EM GP and Titan Krios

Cryo-EM grids were prepared using a Leica EM GP automatic plunge freezer (Leica Microsystems) with 80% humidity at 20°C. 3 µL aliquots were applied onto 60 s glow-discharged, 300 mesh, copper Quantifoil grids (R2/1). After blotting, grids were plunge frozen in liquid ethane cooled by liquid nitrogen.
Micrographs were acquired 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 zero loss energy window; Gatan Inc). Images were recorded 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 pixel sizes of 0.831 Å or 0.629 Å, and a total dose of ~69 electrons per square Angstrom (e^-/Ų) for each micrograph. Micrographs were processed and analyzed during data collection with FOCUS (Biyani et al., 2017 (link)), applying drift-correction and dose-weighting using MotionCor2 (Zheng et al., 2017 (link)). Specific data collection parameters for the various datasets are detailed in Table 2.