Fig. 5: Structures of the NUB1–FAT10-bound hs26S proteasome.

a, For the cryo-EM sample, the hs26S proteasome was mixed with the preformed NUB1–FAT10–Eos complex and incubated for 30 s before freezing. NUB1–FAT10–Eos-bound proteasome particles were then classified into nonprocessing and processing states. b, Representative states for the nonprocessing proteasome reveal the high flexibility of Rpn1 and consequently the Rpn1-bound NUB1–FAT10–Eos complex. Proteasomes were classified on the basis of Rpn1 conformations, showing a continuum of dynamic and more rigid states. This classification yielded a high-resolution reconstruction for the entire 19S regulatory particle (RP), Rpn1 (red) and the Rpn1-bound UBL domain of NUB1 (yellow). c, Left, local refinement of Rpn1 in the nonprocessing hs26S proteasome shows the NUB1 UBL domain (yellow) and the neighboring linker (purple) bound to Rpn1 (red). Right, local refinement of the Rpn1–NUB1^UBL portion of the nonprocessing hs26S proteasome particles allows unambiguous atomic modeling, showing the interactions of a β-sheet in the NUB1 UBL domain (yellow) with the T2 site of Rpn1 (red) and the docking of F169 in the NUB1 UBL linker (purple) with a hydrophobic pocket of Rpn1. d, Low-threshold representation revealing an amorphous, poorly resolved density (purple) that likely represents the flexible core body of NUB1 with bound FAT10–Eos. e, Low-pass-filtered (8 Å) cryo-EM map of the substrate-processing hs26S proteasome that was established by incubating proteasomes with NUB1 and FAT10–Eos for 60 s before freezing. Although highly dynamic and poorly resolved, NUB1 (purple) is still bound at this stage of substrate processing. A more distinct yet low-resolution density (green) at the entrance of the ATPase N-ring potentially represents the tough-to-unfold Eos moiety of the FAT10–Eos substrate after the FAT10 portion has been unfolded and translocated into the central channel. f, Nonfiltered density of the substrate-processing hs26S proteasome, rotated relative to the depiction in e by ~90° to the left and with the ATPase subunits Rpt4 and Rpt5 removed for a better view of the central channel. Substrate density continues through the AAA+ motor channel and into the 20S core particle (CP), indicating that likely the entire FAT10 portion of the FAT10–Eos substrate was unfolded and translocated at this stage of substrate degradation.