Fig. 1: Reconstitution and cryo-EM analysis of the human 26S proteasome in complex with a K11/K48-branched Ub chain-modified Sic1^PY.

a Construct design and labeling strategy of a polyubiquitinated Sic1^PY. b 26S proteasome-mediated proteolysis of the polyubiquitinated Sic1^PY detected by SDS-PAGE separation followed by fluorescence imaging of the exogenously labeled AlexaFluor647. c Evidence of Ub chain branching by Lb^pro* digestion followed by intact MS analysis. The difference of 114 Da is indicative of a single ubiquitination site on the target Ub. The relative populations in percentage are derived from peak integration of the individual peaks. The experiments were carried in triplicate with the original data points shown in open circles and the error bars indicating the standard deviations. d MS Ub-AQUA/PRM quantification of the abundances of the individual Ub chain linkage types. K63 is omitted due to the use of the Ub(K63R) variant. The experiments were carried in triplicate with the original data points shown in open circles and the error bars indicating the standard deviations. Cryo-EM maps of the reconstituted proteasomal complex in the E_B state (e), and substrate-processing E_D state (f), Well-resolved cryo-EM map of the K11/K48-branched Ub₄ chain can be identified in both states. g Segmented cryo-EM map of the proteasome-bound Ub₄ in the E_D state is superimposed with the atomic model to illustrate the clear definition of the K11/K48-branched Ub chain topology. The positions of the Ub_I, Ub_II, Ub_III and Ub_IV are indicated in Roman numbers. The Ub_II density was lowpass filtered to enhance the readability.