Tecnai polara electron microscope

Freshly purified APC/C samples were first visualized by negative-staining EM to check the sample quality and homogeneity and to get initial low-resolution reconstructions. Micrographs were recorded on an FEI Spirit electron microscope at an accelerating voltage of 120 kV and at a defocus of approximately -1.5 µm. For cryo-EM, 2 µl aliquots of the sample at ~0.15 mg/ml were applied onto the Quantifoil R2/2 grids coated with a layer of continuous carbon film (approximately 50 Å thick). Grids were treated with a 9:1 argon:oxygen plasma cleaner for 20 to 40 s before use. The grids were incubated for 30 s at 4°C and 100% humidity before blotting for 5 s and plunging into liquid ethane using an FEI Vitrobot III. The grids were loaded into an FEI Tecnai Polara electron microscope at an acceleration voltage of 300 kV. Micrographs were taken using EPU software (FEI) at a nominal magnification of 78,000 which yields a pixel size of 1.36 Å/pixel. They were recorded by an FEI Falcon III direct electron detector with a defocus range of -2.0 to -4.0 µm. The exposure time for each micrograph was 2s at a dose rate of 27 electrons/Ų/s. 34 movie frames were recorded for each micrograph as described33 (link).