Fig. 1. Time-resolved cryo-EM analysis of the conformational landscape of USP14–proteasome complexes in the act of substrate degradation.

a, b, Cryo-EM density map of the substrate-engaged USP14–proteasome complex in state ${\mathrm{E}}_{\mathrm{D2}.1}^{\mathrm{USP14}}$, viewed from the top (a) and side (b). c, Side view of the cryo-EM density map of the substrate-engaged USP14–proteasome complex in state ${\mathrm{E}}_{\mathrm{D4}}^{\mathrm{USP14}}$. Compared to the view of ${\mathrm{E}}_{\mathrm{D2}.1}^{\mathrm{USP14}}$ in b, USP14 is rotated about 30° to dock onto the AAA domain of RPT1. To visualize the substrate density inside the AAA-ATPase motor, the density of RPT5 is omitted in both b and c. d, Atomic model of state ${\mathrm{E}}_{\mathrm{D2}.1}^{\mathrm{USP14}}$ viewed from the same perspective as in a. e, Kinetic changes of overall particle populations of S_D-like and E_D-like states versus E_A-like states obtained from time-resolved cryo-EM analysis. E_A-like states include ${\mathrm{E}}_{\mathrm{A1}}^{\mathrm{UBL}}$, ${\mathrm{E}}_{\mathrm{A2}.0}^{\mathrm{UBL}}$ and ${\mathrm{E}}_{\mathrm{A2}.1}^{\mathrm{UBL}}$. S_D-like states include ${\mathrm{S}}_{\mathrm{B}}^{\mathrm{USP14}}$, ${\mathrm{S}}_{\mathrm{C}}^{\mathrm{USP14}}$ ${\mathrm{S}}_{\mathrm{D4}}^{\mathrm{USP14}}$ and ${\mathrm{S}}_{\mathrm{D5}}^{\mathrm{USP14}}$. E_D-like states include ${\mathrm{E}}_{\mathrm{D4}}^{\mathrm{USP14}}$, ${\mathrm{E}}_{\mathrm{D5}}^{\mathrm{USP14}}$, ${\mathrm{E}}_{\mathrm{D0}}^{\mathrm{USP14}}$, ${\mathrm{E}}_{\mathrm{D1}}^{\mathrm{USP14}}$, ${\mathrm{E}}_{\mathrm{D2}.0}^{\mathrm{USP14}}$ and ${\mathrm{E}}_{\mathrm{D2}.1}^{\mathrm{USP14}}$. The control consists of previously reported data for substrate-free, USP14-free proteasome⁸. f, Kinetic changes of the particle populations of 13 coexisting conformational states of USP14-bound proteasome from the cryo-EM samples made at different time points after mixing the substrate with the USP14–proteasome complex in the presence of 1 mM ATP at 10 °C. Three substrate-inhibited intermediates (${\mathrm{S}}_{\mathrm{B}}^{\mathrm{USP14}}$, ${\mathrm{S}}_{\mathrm{C}}^{\mathrm{USP14}}$ and ${\mathrm{S}}_{\mathrm{D4}}^{\mathrm{USP14}}$) reach their maximal populations at around 5 min, in contrast to state ${\mathrm{S}}_{\mathrm{D5}}^{\mathrm{USP14}}$ and six substrate-engaged states, which all reach their maximal populations at approximately 1 min. The number of particles used in e and f are provided in Extended Data Fig. 2b, c.