High-resolution cryo-EM data were acquired on Titan Krios operated at 300 keV with a FalconIII detector in counting mode with 20 frames per movie and nominal magnification of 96000× (0.845 Å pixel size). The electron dose was 33.6 e/Å2. For the sprayed apoferritin dataset the defocus range was −0.5 to −3.5 μm, while the vitrobot apoferritin dataset had a range of −0.5 to −2.5 μm. The larger defocus was due to the slightly thicker ice in sprayed samples. Movies were motion-corrected by using MotionCor2 with 5 by 5 patches76 (link) and CTF estimation was done by CTFfind4 on dose-weighted micrographs77 (link). All particle picking was done by crYOLO using a model trained in-house78 (link). Subsequent image processing was performed in Scipion and RELION-3, using PDB 1AEW79 (link), filtered to 40 Å as a reference80 (link),81 (link). CSN5H138A-SCF-N8Skp2/Cks1 complexes were purified as described39 ,82 (link), and data for 2D classification collected on a Talos Arctica (Thermo-Fisher Scientific, Waltham, MA) operating at 200 keV and with a Falcon III direct electron detector in linear mode with 10 frames per movie, 6.5 e/Å2, 1.61 Å pixel size. Movies were aligned using MotionCor2 and CTF estimation was done by gCTF83 (link). Particle picking was done by crYOLO using an in-house trained model78 (link) followed by 2D classification in cryoSPARC 2.14.284 (link).
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