Fig. 4: Visualizing a partially unfolded Eos intermediate during active degradation by the 26S proteasome.

a, Atomic model of the human proteasome in the PS_Rpt2 state during degradation of the FAT10–Eos model substrate. EM density (green) is shown for the substrate, with the partially unraveled β-barrel of Eos pulled against Rpn11 and a translocating polypeptide spanning the central channel of the ATPase motor. b, Focus on the Rpt hexamer with the EM density and atomic model (green) shown for the translocating substrate, which is engaged by a staircase of ATPase domains. The substrate density is at high enough resolution to identify the C-terminal portion of FAT10 inside the ATPase ring. c, Comparison between the partially unfolded Eos intermediate (left, green) and the crystal structure of folded Eos (right, cyan; PDB 3S05), with the chromophore shown in yellow and dark blue, respectively. The intermediate has β1 and the β2–β3 and β5–β6 hairpins partially pulled off from the β-barrel. d, EM density and atomic model for the chromophore in partially unfolded Eos. e, Overlay of the folded (cyan) and partially unfolded Eos (green) show the disruption of the chromophore environment that likely leads to a loss of fluorescence. f, Hydrophobic residues in β1 and the β5–β6 hairpin that normally face the Eos hydrophobic core interact with Rpn11 near the catalytic groove and the Ins-1 loop. g, EM density for proteasomes with bound TXNL1 and the Eos unfolding intermediate show an overlap of binding sites for Eos and TXNL1’s C-terminal tail on Rpn11.