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Development of “Plug and Play” Fiducial Marks for Structural Studies of GPCR Signaling Complexes by Single-Particle Cryo-EM

Dutka, Przemyslaw and Mukherjee, Somnath and Gao, Xiang and Kang, Yanyong and de Waal, Parker W. and Wang, Lei and Zhuang, Youwen and Melcher, Karsten and Zhang, Cheng and Xu, H. Eric and Kossiakoff, Anthony A. (2019) Development of “Plug and Play” Fiducial Marks for Structural Studies of GPCR Signaling Complexes by Single-Particle Cryo-EM. Structure, 27 (12). pp. 1862-1874. ISSN 0969-2126. doi:10.1016/j.str.2019.10.004.

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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 G_i and G_s, as well as mini-G_s, 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 G_i and G_s, 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.

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Zhang, Cheng0000-0001-8288-5823
Additional Information:© 2019 Elsevier Ltd. Received 14 June 2019, Revised 26 August 2019, Accepted 7 October 2019, Available online 25 October 2019. We thank Shohei Koide for providing the DNA of phage Library E and Satchal K. Erramilli for helpful discussion. This work was supported by NIH grants GM117372 (to A.A.K.), GM127710 (to H.E.X.) and 1R35GM128641-01 (to C.Z.). We also acknowledge support from Pfizer (to A.A.K.) and Jay and Betty Van Andel Foundation (to H.E.X.). Data and Code Availability: Molecular dynamics simulation system parameters and trajectories are available upon request. Author Contributions: Conceptualization, P.D., S.M., H.E.X., and A.A.K.; Methodology, P.D. and S.M.; Investigation, P.D., S.M., X.G., Y.K., P.W.d.W., L.W., and Y.Z.; Writing – Original Draft, P.D., S.M., and A.A.K.; Writing – Review & Editing, P.D., S.M., and A.A.K.; Visualization, P.D.; Supervision, K.M., C.Z., H.E.X., and A.A.K.; Funding Acquisition, C.Z., H.E.X., and A.A.K. The authors declare no competing interests.
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Jay and Betty Van Andel FoundationUNSPECIFIED
Subject Keywords:phage display; synthetic antibodies; GPCR; G proteins; GRK (G protein receptor kinase); SP cryo-EM; universal fiducial mark; interface energetics; affinity maturation
Issue or Number:12
Record Number:CaltechAUTHORS:20191223-145547096
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Official Citation:Przemyslaw Dutka, Somnath Mukherjee, Xiang Gao, Yanyong Kang, Parker W. de Waal, Lei Wang, Youwen Zhuang, Karsten Melcher, Cheng Zhang, H. Eric Xu, Anthony A. Kossiakoff, Development of “Plug and Play” Fiducial Marks for Structural Studies of GPCR Signaling Complexes by Single-Particle Cryo-EM, Structure, Volume 27, Issue 12, 2019, Pages 1862-1874.e7, ISSN 0969-2126, (
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:100419
Deposited By: Tony Diaz
Deposited On:23 Dec 2019 23:20
Last Modified:16 Nov 2021 17:53

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