7NAO

Human PA28-20S proteasome complex

Summary for 7NAO

• Entry DOI: 10.2210/pdb7nao/pdb
• EMDB information: 24275 24276 24277 24278
• Descriptor: Proteasome subunit alpha type-2, Proteasome subunit beta type-2, Proteasome subunit beta type-5, ... (16 entities in total)
• Functional Keywords: proteasome, 20s, pa28, hydrolase
• Biological source: Homo sapiens (Human); More
• Total number of polymer chains: 35
• Total formula weight: 957654.25

Authors

Zhao, J.,Makhija, S.,Huang, B.,Cheng, Y. (deposition date: 2021-06-22, release date: 2022-11-02, Last modification date: 2024-06-05)

Primary citation

Zhao, J.,Makhija, S.,Zhou, C.,Zhang, H.,Wang, Y.,Muralidharan, M.,Huang, B.,Cheng, Y.
Structural insights into the human PA28-20S proteasome enabled by efficient tagging and purification of endogenous proteins.
Proc.Natl.Acad.Sci.USA, 119:e2207200119-e2207200119, 2022

PubMed Abstract: The ability to produce folded and functional proteins is a necessity for structural biology and many other biological sciences. This task is particularly challenging for numerous biomedically important targets in human cells, including membrane proteins and large macromolecular assemblies, hampering mechanistic studies and drug development efforts. Here we describe a method combining CRISPR-Cas gene editing and fluorescence-activated cell sorting to rapidly tag and purify endogenous proteins in HEK cells for structural characterization. We applied this approach to study the human proteasome from HEK cells and rapidly determined cryogenic electron microscopy structures of major proteasomal complexes, including a high-resolution structure of intact human PA28αβ-20S. Our structures reveal that PA28 with a subunit stoichiometry of 3α/4β engages tightly with the 20S proteasome. Addition of a hydrophilic peptide shows that polypeptides entering through PA28 are held in the antechamber of 20S prior to degradation in the proteolytic chamber. This study provides critical insights into an important proteasome complex and demonstrates key methodologies for the tagging of proteins from endogenous sources.

PubMed: 35858375
DOI: 10.1073/pnas.2207200119

Experimental method

ELECTRON MICROSCOPY (2.9 Å)