期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：33
DOI：10.1073/pnas.2207200119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
We report an approach of combining CRISPR-Cas9 gene editing with florescence cell sorting to tag and purify human endogenous protein complexes from HEK cells for structural studies by single-particle cryogenic electron microscopy. This procedure is demonstrated by applying the method to study human proteasomal complexes, enabling rapid determination of high-resolution structures of several proteasomal complexes. We envision this approach will enable structural, biochemical, and biophysical studies of many important endogenous human macromolecular complexes.
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.
关键词：ensingle-particle cryo-EMproteasomeendogenous protein taggingCRISPR-Cas9
