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...

Descripción completa

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
_version_ 1783639141347491840
author Chen, Xiang
Shi, Dan
Zhang, Ping
Walters, Kylie J.
author_facet Chen, Xiang
Shi, Dan
Zhang, Ping
Walters, Kylie J.
author_sort Chen, Xiang
collection PubMed
description 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).
format Online
Article
Text
id pubmed-7820132
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher Elsevier
record_format MEDLINE/PubMed
spelling pubmed-78201322021-01-29 An optimized protocol for acquiring and processing cryo-EM data of human 26S proteasome with M1-Ub(6) Chen, Xiang Shi, Dan Zhang, Ping Walters, Kylie J. STAR Protoc Protocol 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). Elsevier 2021-01-18 /pmc/articles/PMC7820132/ /pubmed/33521680 http://dx.doi.org/10.1016/j.xpro.2020.100278 Text en http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Chen, Xiang
Shi, Dan
Zhang, Ping
Walters, Kylie J.
An optimized protocol for acquiring and processing cryo-EM data of human 26S proteasome with M1-Ub(6)
title An optimized protocol for acquiring and processing cryo-EM data of human 26S proteasome with M1-Ub(6)
title_full An optimized protocol for acquiring and processing cryo-EM data of human 26S proteasome with M1-Ub(6)
title_fullStr An optimized protocol for acquiring and processing cryo-EM data of human 26S proteasome with M1-Ub(6)
title_full_unstemmed An optimized protocol for acquiring and processing cryo-EM data of human 26S proteasome with M1-Ub(6)
title_short An optimized protocol for acquiring and processing cryo-EM data of human 26S proteasome with M1-Ub(6)
title_sort optimized protocol for acquiring and processing cryo-em data of human 26s proteasome with m1-ub(6)
topic Protocol
url 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
work_keys_str_mv AT chenxiang anoptimizedprotocolforacquiringandprocessingcryoemdataofhuman26sproteasomewithm1ub6
AT shidan anoptimizedprotocolforacquiringandprocessingcryoemdataofhuman26sproteasomewithm1ub6
AT zhangping anoptimizedprotocolforacquiringandprocessingcryoemdataofhuman26sproteasomewithm1ub6
AT walterskyliej anoptimizedprotocolforacquiringandprocessingcryoemdataofhuman26sproteasomewithm1ub6
AT chenxiang optimizedprotocolforacquiringandprocessingcryoemdataofhuman26sproteasomewithm1ub6
AT shidan optimizedprotocolforacquiringandprocessingcryoemdataofhuman26sproteasomewithm1ub6
AT zhangping optimizedprotocolforacquiringandprocessingcryoemdataofhuman26sproteasomewithm1ub6
AT walterskyliej optimizedprotocolforacquiringandprocessingcryoemdataofhuman26sproteasomewithm1ub6

Ejemplares similares