Cryo-EM grid preparation and data collection

Four microliters of dialyzed sample was deposited onto glow-discharged C-flat Au 1.2/1.3 grids (Electron Microscopy Sciences) and flash-frozen using a manual plunger. Grids were screened at the University of Colorado Boulder Cryo-EM facility using a Tecnai T20 microscope with a K3 electron detector. High-quality grids were then sent to the Janelia Research Campus cryo-EM facility for data acquisition. The complex was imaged at a magnification of ×64000 on an FEI Titan Krios (300 kV) equipped with a Gatan K3 summit direct detector. Pixel size was 1.065 Å. The movies were captured in superresolution mode with an electron dose rate at 7.8 electrons per pixel per second for 5.593 s at 0.13 s per frame. The defocus range was −1.2 to −2.5 μm. A total of 11,160 images were collected.