Departments of Medicinal Chemistry and Molecular Pharmacology and of Chemistry, Center for Cancer Research, and Institute for Drug Discovery, Purdue University, 720 Clinic Drive, West Lafayette, IN 47907, USA.
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China.
STAR Protoc. 2022 Apr 22;3(2):101339. doi: 10.1016/j.xpro.2022.101339. eCollection 2022 Jun 17.
Valosin-containing protein (VCP, also known as p97/Cdc48) comprises six identical 97 kDa VCP protomers and functions as a master regulator of cellular homeostasis. VCP dodecamer in an apo nucleotide status was recently reported, providing a new framework for studying VCP's diverse biological functions. Here, we present a detailed protocol for purifying and cryo-EM structurally characterizing VCP dodecamers from both bacterial and mammalian cells. This protocol can also be adapted to yeast Cdc48. For complete details on the use and execution of this protocol, please refer to Yu et al. (2021).
包含缬氨酸的蛋白（VCP，也称为 p97/Cdc48）由六个相同的 97 kDa VCP 原聚体组成，作为细胞内稳态的主要调节剂。最近报道了 apo 核苷酸状态下的 VCP 十二聚体，为研究 VCP 多样化的生物学功能提供了新的框架。在这里，我们提供了一个从细菌和哺乳动物细胞中纯化和低温电镜结构表征 VCP 十二聚体的详细方案。该方案也可以适应酵母 Cdc48。有关此方案的使用和执行的完整详细信息，请参阅 Yu 等人。（2021）。
