An optimized protocol for acquiring and processing cryo-EM data of human 26S proteasome with M1-Ub(6)

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...

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Detalles Bibliográficos
Autores principales: Chen, Xiang, Shi, Dan, Zhang, Ping, Walters, Kylie J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2021
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820132/
https://www.ncbi.nlm.nih.gov/pubmed/33521680
http://dx.doi.org/10.1016/j.xpro.2020.100278
Descripción
Sumario: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-Ub(6)) unanchored to a substrate. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020).

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