Purification and cryo-EM structure determination of VCP/p97 dodecamers from mammalian and bacterial cells
Guimei Yu1,2, Yunpeng Bai1, Zhong-Yin Zhang1
1Departments 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.
Abstract:
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).
