Sample preparation and cryo‐electron microscopy analysis

In order to increase the stability of the 5‐ and 6‐subunit hGID complexes, the gradient fixation (GraFix) protocol was applied (Stark, 2010). Briefly, samples were loaded on a glycerol gradient (10–40% w/v) in the presence of the cross‐linker glutaraldehyde (0.25% v/v added to the 40% glycerol solution), followed by ultracentrifugation (SW40Ti rotor) at 125,750 g for 18 h at 4°C. Peak fractions containing the protein complexes were collected, and buffer exchange for glycerol removal was performed by Zeba Spin columns in a buffer containing 50 mM HEPES pH 7.4, 200 mM NaCl, 1 mM TCEP and either 0.01% NP‐40 for the 5‐subunit hGID complex or 0.05% NP‐40 for the 6‐subunit hGID complex. 4 μl sample (0.08–0.15 mg/ml) was then applied on glow discharged Quantifoil holey grids (R2.2, Cu 300 mesh, Quantifoil Micro Tools GmbH, Grosslöbichau, Germany) coated with a continuous 1 nm carbon film. Grids were incubated for 20–60 s at 4°C and 100% humidity, blotted for 1 s with Whatman no.1 filter paper, and vitrified by plunging into liquid ethane (Vitrobot, Thermo Fischer).

Three datasets of 5‐subunit GID and one dataset of GID‐ARMC8β complexes were collected with the Titan Krios cryo‐electron microscope (Thermo Fisher Scientific Inc., Waltham MA) operated at 300 kV, using the K2 and K3 direct electron detectors (Gatan Inc., Pleasanton CA), operated in counting or super‐resolution mode. Data collection parameters are compiled in Table EV1.