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Development of "Plug and Play" Fiducial Marks for Structural Studies of GPCR Signaling Complexes by Single-Particle Cryo-EM.
Structure ( IF 4.4 ) Pub Date : 2019-10-25 , DOI: 10.1016/j.str.2019.10.004 Przemyslaw Dutka 1 , Somnath Mukherjee 1 , Xiang Gao 2 , Yanyong Kang 2 , Parker W de Waal 2 , Lei Wang 3 , Youwen Zhuang 4 , Karsten Melcher 2 , Cheng Zhang 3 , H Eric Xu 4 , Anthony A Kossiakoff 5
Structure ( IF 4.4 ) Pub Date : 2019-10-25 , DOI: 10.1016/j.str.2019.10.004 Przemyslaw Dutka 1 , Somnath Mukherjee 1 , Xiang Gao 2 , Yanyong Kang 2 , Parker W de Waal 2 , Lei Wang 3 , Youwen Zhuang 4 , Karsten Melcher 2 , Cheng Zhang 3 , H Eric Xu 4 , Anthony A Kossiakoff 5
Affiliation
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"Universal" synthetic antibody (sAB)-based fiducial marks have been generated by customized phage display selections to facilitate the rapid structure determination of G protein-coupled receptor (GPCR) signaling complexes by single-particle cryo-electron microscopy (SP cryo-EM). sABs were generated to the two major G protein subclasses: trimeric Gi and Gs, as well as mini-Gs, and were tested to ensure binding in the context of their cognate GPCRs. Epitope binning revealed that multiple distinct epitopes exist for each G(αβγ) protein. Several Gβγ-specific sABs, cross-reactive between trimeric Gi and Gs, were identified suggesting they could be used across all subclasses in a "plug and play" fashion. sABs were also generated to a representative of another class of GPCR signaling partner, G protein receptor kinase 1 (GRK1) and evaluated further, supporting the generalizability of the approach. EM data suggested that the subclass-specific sABs provide effective single and dual fiducials for multiple GPCR signaling complexes.
中文翻译：
单颗粒Cryo-EM用于GPCR信号复合物结构研究的“即插即用”基准标记的开发。
通过定制的噬菌体展示选择已生成基于“通用”合成抗体（sAB）的基准标记，以利于通过单粒子冷冻电子显微镜（SP cryo-EM）快速确定G蛋白偶联受体（GPCR）信号复合物的结构）。sABs产生于两个主要的G蛋白亚类：三聚Gi和Gs，以及mini-G，并经过测试以确保在其同源GPCR的情况下具有结合力。表位分级显示每个G（αβγ）蛋白存在多个不同的表位。鉴定了在三聚体Gi和Gs之间交叉反应的几种Gβγ特异性sAB，表明它们可以“即插即用”方式用于所有亚类。还向另一类GPCR信号传递伙伴的代表生成了sAB，G蛋白受体激酶1（GRK1）并进行了进一步评估，支持该方法的通用性。EM数据表明，特定于亚类的sAB为多种GPCR信号复合物提供了有效的单基准和双基准。
更新日期：2019-10-25
中文翻译：
单颗粒Cryo-EM用于GPCR信号复合物结构研究的“即插即用”基准标记的开发。
通过定制的噬菌体展示选择已生成基于“通用”合成抗体（sAB）的基准标记，以利于通过单粒子冷冻电子显微镜（SP cryo-EM）快速确定G蛋白偶联受体（GPCR）信号复合物的结构）。sABs产生于两个主要的G蛋白亚类：三聚Gi和Gs，以及mini-G，并经过测试以确保在其同源GPCR的情况下具有结合力。表位分级显示每个G（αβγ）蛋白存在多个不同的表位。鉴定了在三聚体Gi和Gs之间交叉反应的几种Gβγ特异性sAB，表明它们可以“即插即用”方式用于所有亚类。还向另一类GPCR信号传递伙伴的代表生成了sAB，G蛋白受体激酶1（GRK1）并进行了进一步评估，支持该方法的通用性。EM数据表明，特定于亚类的sAB为多种GPCR信号复合物提供了有效的单基准和双基准。
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