Best-scoring configurations of the AAA-ATPase subunits. A: Schematic representation of the AAA-ATPase subunit order and the contact restraints acting on them (Table 1). The gray circles represent subunits with Pro-62 in the cis-conformation. B: One of two possible quaternary structure arrangements of the N- and AAA-ring fitted into the segmented 3-fold symmetric EM map. C: The AAA-ring is rotated by 60° with respect to the N-ring in the second quaternary structure. In the two quaternary structures, a 9-residue linker spans a distance of approximately 21 Å and 26 Å, respectively, both of which are compatible with a typical Cα-Cα distance of 3.5 Å.

Different assembly chaperones are required to position the AAA-ATPases on the CP. A: Model for the assembly process of the 26S proteasome. The assembly consists of at least five different steps: (i) Formation of different base-precursor complexes, each bound to a different chaperone (cyan). (ii) The lid and the remaining base subunits form a sub-complex with the AAA-ATPase pair Rpt3/Rpt6. (iii) An RP precursor lacking only Rpt4/Rpt5 forms. (iv) The Rpt4/Rpt5 dimer completes the RP and the chaperone Nas2 dissociates. (v) The three chaperones Nas6, Rpn14, and Hsm13 dissociate upon binding of RP and CP. B: Quaternary structure of Rpt1 (binds to assembly chaperone Hsm3) and the two adjacent CP alpha subunits (gold). C: Same for Rpt3 (binds to Nas6), and D: Rpt6 (Rpn14). E: Positioning Nas6 (PDB code 2DZN, [36]) on our CP-AAA-ATPase model does not lead to steric clashes with the CP.

Segmentation of the AAA-ATPase from the 26S proteasome map. A. The ATPase density (orange) was segmented from the 26S proteasome map (yellow) based on the approximate 6-fold symmetry of the AAA-ring. Three distinct rod-shaped features protrude from the AAA-ATPase density (red). The displayed map is one of two 26S conformations described in [9], which lacks an additional mass (probably Rpn10). B. Segmented AAA-ATPase using 6-fold symmetry. The rod-like features are not present in the map at the chosen contour level. C. Segmented AAA-ATPase using 3-fold symmetry. At the three corners of the map rod-like features project out of the density (red).

AAA-ATPase-CP model. A: The atomic model of the AAA-ATPase-CP sub-complex fitted into the cryo-EM map of the 26S proteasome. The AAA-ATPase model is placed on the top. The six AAA-ATPase subunits and the adjacent CP α-subunits are colored as indicated in panel C, the remaining CP subunits in gold. The AAA-ATPase quaternary structure corresponds to Fig. 2C. B: Zoomed-in stereo view of the rectangular area in A. C: Schematic view of the topology of the CP-AAA-ATPase model and the CP-RP interactions (Table 1). The AAA-ATPase subunits are displayed in Arabic numbers, whereas Roman numbers indicate the CP α-subunits. The asterisks mark the AAA-ATPase subunits that have been shown to induce gate opening of the CP (Rpt2 and Rpt5).