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Fusion of DARPin to aldolase enables visualization of small protein by cryoEM

Yao, Qing and Weaver, Sara J. and Mock, Jee-Young and Jensen, Grant J. (2018) Fusion of DARPin to aldolase enables visualization of small protein by cryoEM. . (Unpublished) http://resolver.caltech.edu/CaltechAUTHORS:20181107-101625710

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Abstract

In recent years, solving protein structures by single particle cryogenic electron microscopy (cryoEM) has become a crucial tool in structural biology. While exciting progress is being made towards the visualization of smaller and smaller macromolecules, the median protein size in both eukaryotes and bacteria is still beyond the reach of single particle cryoEM. To overcome this problem, we implemented a platform strategy in which a small protei target was rigidly attached to a large, symmetric base via a selectable adapter. Seven designs were tested. In the best construct, a designed ankyrin repeat protein (DARPin) was rigidly fused to tetrameric rabbit muscl aldolase through a helical linker. The DARPin retained its ability to bind its target, the 27 kDa green fluorescent protein (GFP). We solve the structure of this complex to 3.0 Å resolution overall, with 5 to 8 Å resolution in the GFP region. As flexibility in the DARPin limited the overall resolution of the target, we described strategies to rigidify this element.


Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
https://doi.org/10.1101/455063DOIDiscussion Paper
ORCID:
AuthorORCID
Yao, Qing0000-0003-3575-9909
Mock, Jee-Young0000-0002-4656-3357
Jensen, Grant J.0000-0003-1556-4864
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. bioRxiv preprint first posted online Oct. 30, 2018. 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. This work was supported by funds from NIH NIGMS P50 082545. CryoEM work was performed in the Beckman Institute Resource Center for Transmission Electron Microscopy at Caltech. Data deposition: Density map of GFP:DARPin-aldolase complex has been deposited in the Electron Microscopy Data Bank (EMDB) with access code: EMD-9277 and PDB 6MWQ.
Funders:
Funding AgencyGrant Number
NIHP50 082545
Record Number:CaltechAUTHORS:20181107-101625710
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20181107-101625710
Official Citation:Fusion of DARPin to aldolase enables visualization of small protein by cryoEM. Qing Yao, Sara J Weaver, Jee-Young Mock, Grant J Jensen. bioRxiv 455063; doi: https://doi.org/10.1101/455063
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:07 Nov 2018 19:28

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