| Autor: |
Cooney, Ian, Mack, Deirdre C., Ferrell, Aaron J., Stewart, Michael G., Wang, Shuxin, Donelick, Helen M., Tamayo-Jaramillo, Daniela, Greer, Dakota L., Zhu, Danyang, Li, Wenyan, Shen, Peter S. |
| Rok vydání: |
2023 |
| Předmět: |
|
| Zdroj: |
Bio Protoc |
| ISSN: |
2331-8325 |
| Popis: |
Single-particle electron cryo-microscopy (cryo-EM) is an effective tool to determine high-resolution structures of macromolecular complexes. Its lower requirements for sample concentration and purity make it an accessible method to determine structures of low-abundant protein complexes, such as those isolated from native sources. While there are many approaches to protein purification for cryo-EM, attaining suitable particle quality and abundance is generally the major bottleneck to the typical single-particle project workflow. Here, we present a protocol using budding yeast ( S. cerevisiae ), in which a tractable immunoprecipitation tag (3xFLAG) is appended at the endogenous locus of a gene of interest (GOI). The modified gene is expressed under its endogenous promoter, and cells are grown and harvested using standard procedures. Our protocol describes the steps in which the tagged proteins and their associated complexes are isolated within three hours of thawing cell lysates, after which the recovered proteins are used directly for cryo-EM specimen preparation. The prioritization of speed maximizes the ability to recover intact, scarce complexes. The protocol is generalizable to soluble yeast proteins that tolerate C-terminal epitope tags. Graphical abstract [Image: see text] Overview of lysate-to-grid workflow. Yeast cells are transformed to express a tractable tag on a gene of interest. Following cell culture and lysis, particles of interest are rapidly isolated by co-immunoprecipitation and prepared for cryo-EM imaging (created with BioRender.com). |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
|
