Vitrobot 3

A 4 μl aliquot of full-length BAM complex in 20 mM Tris pH 8.0, 150 mM NaCl and 0.05% (w/v) n-dodecyl β-d-maltopyranoside (DDM), at a concentration of 7 mg ml⁻¹ with a 10-fold molar excess of dynobactin A was applied to a Quantifoil R2/1 holey carbon copper grid (Quantifoil Micro Tools) vitrified by plunging into liquid ethane using a Vitrobot (FEI, Vitrobot III). One dataset was collected using a Titan Krios electron microscope (FEI) operated at 300 kV, a GIF Quantum LS imaging filter (Gatan) and a K2 Summit (Gatan) operating in counting mode using SerialEM^{75 (link)}. Images were acquired at 0.8–3.0 μm defocus and a nominal magnification of ×165,000, corresponding to a pixel size of 0.82 Å (Supplementary Table 5). Movies were collected with a total dose of approximately 48 e⁻ Å⁻² per 9 s exposure and fractionated over 40 frames using beam-image shift to record 4 holes at 8 images per hole.

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).