An optimized protocol for acquiring and processing cryo-EM data of human 26S proteasome with M1-Ub₆

The 26S proteasome is specialized for regulated protein degradation. It is formed by a regulatory particle (RP) that recognizes ubiquitinated substrates and caps a hollow cylindrical core particle (CP) where substrates are proteolyzed. Structural heterogeneity caused by dynamics makes it challenging to observe ubiquitin chains at the RP by cryogenic electron microscopy (cryo-EM). Here, we present a cryo-EM-based protocol we applied to study the human 26S proteasome with ubiquitin chains by using non-cleavable M1-linked hexaubiquitin (M1-Ub₆) unanchored to a substrate. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020).