Cryogenic Electron Microscopy of Purified PfGCC

To prepare cryoEM grids, we applied 2 μl of purified PfGCC sample to thin continuous carbon films on lacey grids (Ted Pella Inc.) for 1 min, blotted for 12 s under 100% humidity and plunged the grid into liquid ethane with an FEI Vitrobot (ambient temperature in the Vitrobot chamber was 20 °C). CryoEM images were collected at liquid nitrogen temperature in an FEI Titan Krios cryo electron microscope operated at 300 kV using parallel illumination. Before data collection, the microscope was carefully aligned, and beam tilt was minimized by coma-free alignment. Images were recorded on a Gatan K2 camera with the counting mode at a nominal magnification of × 29,000 on microscope. The magnification on the camera was calibrated as × 49,500 using a catalase crystal sample, giving a pixel size of 1.01 Å per pixel on specimen. The dose rate of the electron beam was set to ∼8 counts per pixel per s on the camera which corresponds to ∼10 e⁻ Å⁻² s⁻¹ on specimen when including the electrons uncounted by the K2 camera. Image stacks were recorded at 4 frames per sec for 10 s. After drift correction with the UCSF software34 (link), the first 12 frames of each image stack (movie) were merged to generate a final image with a total dose of ∼30 e⁻ Å⁻² of the sample.