PT - JOURNAL ARTICLE AU - Zhao, Jianhua AU - Makhija, Suraj AU - Huang, Bo AU - Cheng, Yifan TI - Efficient tagging and purification of endogenous proteins for structural studies by single particle cryo-EM AID - 10.1101/2021.06.25.449985 DP - 2021 Jan 01 TA - bioRxiv PG - 2021.06.25.449985 4099 - http://biorxiv.org/content/early/2021/07/02/2021.06.25.449985.short 4100 - http://biorxiv.org/content/early/2021/07/02/2021.06.25.449985.full AB - A major bottleneck in structural biology is producing biologically relevant samples at sufficient quantities. This is particularly true for large protein assemblies where conventional techniques of gene overexpression require substantial optimization, hampering structural studies and drug development efforts. Here we describe a method combining CRISPR/Cas gene editing and fluorescence cell sorting to rapidly tag and purify endogenous human proteins from cell lines, enabling structural analysis of native proteins that are properly folded and assembled. We applied this approach to study the human proteasome from HEK cells and rapidly determined structures of major proteasomal complexes. Structures of the PA28-20S complex reveal the native subunit stoichiometry of PA28 and a distinct functional state of the complex. The efficient strategy for tagging and extracting endogenous proteins described here will enable the structural study of many challenging targets and provide more biologically relevant samples for research and therapeutic development.Competing Interest StatementThe authors have declared no competing interest.