Extended Data Fig. 4: Key structural features that differentiate the seven conformational states.

a, Ubiquitin densities in states E_A1 (left) and E_A2 (right). The T1 site is labelled by fitting the yellow cartoon representation of the NMR structure (RCSB Protein Data Bank (PDB) ID 2N3U) of the yeast Rpn1 T1 element in complex with two ubiquitins into our density, showing that the ubiquitin on human RPN1 is bound to a site very close to the yeast Rpn1 T1 site⁶. The density maps are low-pass-filtered to 8 Å to show the ubiquitin features clearly, owing to the lower local resolution of the ubiquitin density in these maps. b, The ubiquitin–RPN11–RPT5 interface observed at high resolution in state E_B is also observed in state E_A2, albeit at lower local resolution. The E_A2 density is shown as a transparent surface. c, Comparison of the Ins1 loop of RPN11 in different states. d, Comparison of the RPN11 structures in states E_A2, E_B and E_C1 around the zinc-binding site and Ins1 region with that in the crystal structure (PDB ID: 5U4P) of a ubiquitin-bound Rpn11–Rpn8 complex from yeast²⁹. e, Close-up comparison of the RPN11 Ins1 structure between state E_B and 5U4P (left two panels) and between state E_C1 and 5U4P (right two panels) in two orthogonal perspectives, showing a 5 Å displacement of the Ins1 β-hairpin in E_B relative to 5U4P or E_C1. This displacement is not observed between E_C1 and 5U4P, suggesting that the Ins1 β-hairpin tilt in E_B is mostly to optimize the coordination of the isopeptide bond with the zinc ion. f, Comparison of the RP structures of E_A and E_B. g, Comparison of lid subcomplex conformations among all states. h, Comparison of ATPase ring structures between two successive states. The structures are aligned together against their CP in f–h. i, Side views of the structural comparison of the AAA ring between E_C1 (colour) and E_B (grey). The large AAA subdomain of RPT1 was used to align the two AAA-ring structures together. A 40° out-of-plane rotation of the large AAA subdomain of RPT1 relative to the AAA ring is observed during disengagement of RPT1–RPT2 from the substrate. The right panel, rotated vertically against the left panel, shows that the out-of-plane rotation in RPT1 is more substantially amplified in its anticlockwise neighbouring ATPases than in its clockwise neighbours. Red arrows mark the centre of the AAA ring. j, Structural comparison between E_D1 (colour) and E_C2 (grey) in which the large AAA subdomain of RPT1 is used to align the two AAA-ring structures. A small 5° out-of-plane rotation of the large AAA subdomain of RPT1 relative to the AAA ring is observed during the re-engagement of RPT1–RPT2 with the substrate. k, Structural comparison between E_D1 (colour) and E_C2 (grey), by using the large AAA subdomain of RPT5 to align the two AAA-ring structures. A 30° out-of-plane rotation of the large AAA subdomain of RPT5 relative to the AAA ring is observed during disengagement of RPT5 from the substrate. The right panel, rotated vertically against the left panel, shows that the out-of-plane rotation in RPT5 is amplified in its anticlockwise neighbouring ATPases more substantially than in its clockwise neighbours. Red arrows mark the centre of the AAA ring. l, Structural comparison between E_D2 (colour) and E_D1 (grey) in which the large AAA subdomain of RPT5 is used to align the two AAA-ring structures. An 8° out-of-plane rotation of the large AAA subdomain of RPT5 relative to the AAA ring is observed during re-engagement of RPT5 with the substrate.