Extended Data Fig. 1. Protein purification and cryo-EM imaging.

a, The human 26S proteasome was purified through gel-filtration column (Superose 6 10/300 GL). b, Native gel analysis of the human 26S proteasome from (a). c, FPLC purification of human USP14 on Superdex 75 10/300 GL column. d–f, SDS-PAGE and Coomassie blue stain analysis of purified USP14 (d), Sic1^PY, UBE1, UBCH5A, WW-HECT, human RPN13 (e), USP14 UBL and USP domains (f). g and h, Western blot was used to evaluate the content of RPN13 (g) and USP14 (h) in the purified human proteasomes. The results indicate the presence of RPN13 and the absence of USP14 in the purified human 26S proteasome. i, Western blot was used to verify polyubiquitylation of Sic1^PY (Ub_n-Sic1^PY) using anti-T7 antibody, indicating that most Sic1^PY was ubiquitylated. j, In vitro degradation of Ub_n-Sic1^PY by the purified 26S proteasome at 10 °C in the absence and presence of USP14. The concentration and ratio of each component was same as cryo-EM sample preparation. The experiments were repeated three times. Samples in (i) and (j) were analyzed by SDS–PAGE/Western blot using anti-T7 antibody. W/O, the proteasome without binding to USP14. WT, the wildtype USP14-bound proteasome. k, Kinetics of Ub_n-Sic1^PY degradation was plotted by measuring Ub_n-Sic1^PY density in (j) using ImageJ software. Each point is representative of three independent experiments. Data are presented as mean ± s.d. l–o, MST analysis of USP14 binding to the human proteasome. A dissociation constant of 94.6 ± 27.1 nM (l, full-length USP14 in the absence of Ub_n-Sic1^PY), 137 ± 33.4 nM (m, USP14 UBL domain only), 135 ± 22.9 nM (n, USP14 USP domain only) and 43.9 ± 24.7 nM (o, full-length USP14 in the presence of Ub_n-Sic1^PY) were calculated from three independent experiments (shown as mean ± s.d.). p and q, Typical motion-corrected cryo-EM micrographs (left) of the substrate-engaged human USP14–proteasome complex in the presence of 1 mM ATP (p) or after ATP-to-ATPγS exchange (q). Power spectrum evaluation of the corresponding micrographs are shown on the right. The exact numbers of micrographs collected under different experimental conditions are provided in Extended Data Fig. 2a. Each experiment was repeated independently at least five times with similar results. r, Comparison of percent population of each conformational state in the presence of 1 mM ATP or ATP-to-ATPγS exchange in 1 min after mixing the substrate with the USP14-bound proteasome. Our cryo-EM analysis suggests that ATP-to-ATPγS exchange enriches states ${\mathrm{E}}_{\mathrm{A2}.1}^{\mathrm{UBL}}$, ${\mathrm{S}}_{\mathrm{C}}^{\mathrm{USP14}}$, ${\mathrm{S}}_{\mathrm{D5}}^{\mathrm{USP14}}$, ${\mathrm{E}}_{\mathrm{D1}}^{\mathrm{USP14}}$, ${\mathrm{E}}_{\mathrm{D2}.1}^{\mathrm{USP14}}$ and ${\mathrm{E}}_{\mathrm{D4}}^{\mathrm{USP14}}$, but reduces states ${\mathrm{E}}_{\mathrm{A1}}^{\mathrm{UBL}}$, ${\mathrm{E}}_{\mathrm{A2}.0}^{\mathrm{UBL}}$, ${\mathrm{S}}_{\mathrm{B}}^{\mathrm{USP14}}$ and ${\mathrm{E}}_{\mathrm{D2}.0}^{\mathrm{USP14}}$. The particle numbers used to derive this plot are provided in Extended Data Fig. 2c. For gel source data, see Supplementary Fig. 1

Source data