Extended Data Fig. 10: Comparisons of the six discrete substrate-processing states for the human 26S proteasome regarding spiral-staircase arrangements, pore-loop interactions with the substrate polypeptide, nucleotide occupancies, and asymmetric particle distributions.
From: Structural landscape of the degrading 26S proteasome reveals conformation-specific binding of TXNL1
a) Top: Cryo-EM densities of the AAA+ motor for all processing-state (PS) conformation, colored by Rpt subunit. Substrate-disengaged seam subunits are at lower resolution likely due to higher mobility and various vertical positions between the bottom and top of the spiral staircases. PSRpt3, PSRpt6, and PSRpt2 show larger density gaps between subunits at the seam, likely because they contain two substrate-disengaged seam subunits with potentially high continuous motions. Bottom: Spiral staircase arrangements of pore-1-loop Tyr residues (Phe for Rpt5) for each processing state. Distances between the substrate backbone and the backbone Cα of the pore-1 loop aromatic residue are indicated for the disengaged seam subunits. Interestingly, some seam subunits reside at the bottom, while others are observed toward the top of the staircase. Positions of the pore-loop residues for these seam subunits are approximate due to the higher mobility and consequently lower resolution. b) Cryo-EM densities and atomic models showing the ATP binding sites of all Rpts in the six processing states. Images are focused on the subunit interfaces, with the nucleotide-binding residues of a respective Rpt to the right of the nucleotide and interacting residues of the clockwise-next subunit, including Arg fingers, on the left. The nucleoside portions are shown in green for ADP and red for ATP, the phosphodiesters are depicted in orange and red, and the Mg2+ as a green sphere. c) Determining the identity of the nucleotide bound to Rpt3 in PSRpt5 and PSRpt2 was more ambiguous due to lower-resolution, but the observed densities fit ADP-Mg2+ (left) better than ATP-Mg2+ (right). d) Particle numbers observed for each processing state in the separate cryo-EM data sets for the 26S proteasome in the presence of sub-stoichiometric TXNL1, 30 s after incubations with the FAT10 substrate (top) or in the presence of excess TXNL1, 2 min after incubation with FAT10 (bottom). e) Plotted distances between the substrate backbone and the backbone Cα of the pore-1 loop aromatic residue for Rpt1-6 in each substrate-processing state of the proteasome.
