Fusion of DARPin to Aldolase Enables Visualization of Small Protein by Cryo-EM
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.
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.Attached Files
Accepted Version - nihms-1528032.pdf
Submitted - 455063.full.pdf
Supplemental Material - 1-s2.0-S096921261930125X-mmc1.pdf
Supplemental Material - 1-s2.0-S096921261930125X-mmc5.pdf
Files
Additional details
- Alternative title
- Fusion of DARPin to aldolase enables visualization of small protein by cryoEM
- PMCID
- PMC6610650
- Eprint ID
- 90702
- Resolver ID
- CaltechAUTHORS:20181107-101625710
- P50 082545
- NIH
- Caltech Beckman Institute
- Created
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2018-11-07Created from EPrint's datestamp field
- Updated
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2023-06-01Created from EPrint's last_modified field