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Fusion of DARPin to Aldolase Enables Visualization of Small Protein by Cryo-EM

Yao, Qing and Weaver, Sara J. and Mock, Jee-Young and Jensen, Grant J. (2019) Fusion of DARPin to Aldolase Enables Visualization of Small Protein by Cryo-EM. Structure, 27 (7). pp. 1148-1155. ISSN 0969-2126. PMCID PMC6610650. https://resolver.caltech.edu/CaltechAUTHORS:20181107-101625710

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Abstract

Solving protein structures by single-particle cryoelectron microscopy (cryo-EM) has become a crucial tool in structural biology. While exciting progress is being made toward the visualization of small macromolecules, the median protein size in both eukaryotes and bacteria is still beyond the reach of cryo-EM. To overcome this problem, we implemented a platform strategy in which a small protein target was rigidly attached to a large, symmetric base via a selectable adapter. Of our seven designs, the best construct used a designed ankyrin repeat protein (DARPin) rigidly fused to tetrameric rabbit muscle aldolase through a helical linker. The DARPin retained its ability to bind its target: GFP. We solved the structure of this complex to 3.0 Å resolution overall, with 5–8 Å resolution in the GFP region. As flexibility in the DARPin position limited the overall resolution of the target, we describe strategies to rigidify this element.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/j.str.2019.04.003DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610650PubMed CentralArticle
https://doi.org/10.1101/455063DOIDiscussion Paper
ORCID:
AuthorORCID
Yao, Qing0000-0003-3575-9909
Weaver, Sara J.0000-0001-7753-6215
Mock, Jee-Young0000-0002-4656-3357
Jensen, Grant J.0000-0003-1556-4864
Alternate Title:Fusion of DARPin to aldolase enables visualization of small protein by cryoEM
Additional Information:© 2019 Elsevier Ltd. Received 2 December 2018, Revised 4 March 2019, Accepted 5 April 2019, Available online 9 May 2019. We thank Dr. Mark Herzik Jr. and Mengyu Wu at The Scripps Research Institute for help with sample preparation. We also thank Dr. Songye Chen, Dr. Andrey Malyutin, Dr. Rebecca Voorhees, and Dr. Bil Clemons at Caltech for technical assistance. We are also grateful to all members of the Jensen laboratory for discussion and technical assistance. In particular, we thank Dr. Lauren Ann Metskas and Dr. Ariana Peck. This work was supported by funds from NIH NIGMS P50 082545. Cryo-EM work was performed in the Caltech Beckman Institute Resource Center for Transmission Electron Microscopy. Author Contributions: Conceptualization, Q.Y., S.J.W., and G.J.J.; Methodology, Q.Y., S.J.W., and J.-Y.M.; Investigation, Q.Y., S.J.W., and J.-Y.M.; Data Curation, S.J.W.; Writing – Original Draft, S.J.W.; Writing – Review & Editing, S.J.W., Q.Y., and G.J.J.; Visualization, S.J.W. and Q.Y.; Supervision, G.J.J.; Funding Acquisition, G.J.J. The authors declare no competing interests.
Funders:
Funding AgencyGrant Number
NIHP50 082545
Caltech Beckman InstituteUNSPECIFIED
Subject Keywords:CryoEM; DARPin; Single-particle analysis; platform; aldolase; artificial protein; Electron cryo-microscopy; cryo-electron microscopy; protein design
Issue or Number:7
PubMed Central ID:PMC6610650
Record Number:CaltechAUTHORS:20181107-101625710
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20181107-101625710
Official Citation:Qing Yao, Sara J. Weaver, Jee-Young Mock, Grant J. Jensen, Fusion of DARPin to Aldolase Enables Visualization of Small Protein by Cryo-EM, Structure, Volume 27, Issue 7, 2019, Pages 1148-1155.e3, ISSN 0969-2126, https://doi.org/10.1016/j.str.2019.04.003. (http://www.sciencedirect.com/science/article/pii/S096921261930125X)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:90702
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:07 Nov 2018 19:28
Last Modified:05 Mar 2020 19:48

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