All data sets were recorded using 300 keV electrons. The γ-secretase, β-galactosidase, and mitoribosome data sets were recorded manually; the complex-I data set was recorded automatically using the EPU software from FEI. For all data sets, fields of views that showed signs of significant drift, charging, or astigmatism were discarded. For the γ-secretase data, this assessment was made after alignment using the algorithm by Li et al. (2013) (link). Movies on the Falcon-II detectors on the Polara and Titan Krios microscopes were intercepted using a system that was developed in-house (Bai et al., 2013 (link)). CTF parameters were estimated using CTFFIND3 (Mindell and Grigorieff, 2003 (link)), and the particles were picked in a semi-automated manner, using EMAN2 (Tang et al., 2007 (link)) for the mitoribosome, and RELION for the three other data sets. Selection of particles for the final 3D reconstruction was performed using reference-free 2D class averaging and 3D classification in RELION (Scheres, 2012 (link)), and the final maps before and after movie processing were calculated using RELION’s 3D auto-refine, followed by automated B-factor sharpening (Rosenthal and Henderson, 2003 (link)) and correction for the MTF of the detector. All resolutions were based on the gold-standard FSC = 0.143 criterion (Scheres and Chen, 2012 (link)), and FSC curves were corrected for the effects of soft masking by high-resolution noise substitution (Chen et al., 2013 (link)). Density figures were made using UCSF Chimera (Pettersen et al., 2004 (link)).
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