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. doi:10.1016/j.str.2019.04.003. 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:

URL	URL Type	Description
https://doi.org/10.1016/j.str.2019.04.003	DOI	Article
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610650	PubMed Central	Article
https://doi.org/10.1101/455063	DOI	Discussion Paper

ORCID:

Author	ORCID
Yao, Qing	0000-0003-3575-9909
Weaver, Sara J.	0000-0001-7753-6215
Mock, Jee-Young	0000-0002-4656-3357
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 Agency	Grant Number
NIH	P50 082545
Caltech Beckman Institute	UNSPECIFIED

Subject Keywords:

CryoEM; DARPin; Single-particle analysis; platform; aldolase; artificial protein; Electron cryo-microscopy; cryo-electron microscopy; protein design

Issue or Number:

PubMed Central ID:

PMC6610650

DOI:

10.1016/j.str.2019.04.003

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.

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:

01 Jun 2023 22:42

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