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Title: Near-atomic cryo-EM imaging of a small protein displayed on a designed scaffolding system

Abstract

Current single-particle cryo-electron microscopy (cryo-EM) techniques can produce images of large protein assemblies and macromolecular complexes at atomic level detail without the need for crystal growth. However, proteins of smaller size, typical of those found throughout the cell, are not presently amenable to detailed structural elucidation by cryo-EM. Here we use protein design to create a modular, symmetrical scaffolding system to make protein molecules of typical size suitable for cryo-EM. Using a rigid continuous alpha helical linker, we connect a small 17-kDa protein (DARPin) to a protein subunit that was designed to self-assemble into a cage with cubic symmetry. We show that the resulting construct is amenable to structural analysis by single-particle cryo-EM, allowing us to identify and solve the structure of the attached small protein at near-atomic detail, ranging from 3.5- to 5-Å resolution. The result demonstrates that proteins considerably smaller than the theoretical limit of 50 kDa for cryo-EM can be visualized clearly when arrayed in a rigid fashion on a symmetric designed protein scaffold. Furthermore, because the amino acid sequence of a DARPin can be chosen to confer tight binding to various other protein or nucleic acid molecules, the system provides a future route for imaging diversemore » macromolecules, potentially broadening the application of cryo-EM to proteins of typical size in the cell.« less

Authors:
; ; ;
Publication Date:
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1423732
Grant/Contract Number:  
FC02-2ER63421
Resource Type:
Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Volume: 115 Journal Issue: 13; Journal ID: ISSN 0027-8424
Publisher:
Proceedings of the National Academy of Sciences
Country of Publication:
United States
Language:
English

Citation Formats

Liu, Yuxi, Gonen, Shane, Gonen, Tamir, and Yeates, Todd O. Near-atomic cryo-EM imaging of a small protein displayed on a designed scaffolding system. United States: N. p., 2018. Web. doi:10.1073/pnas.1718825115.
Liu, Yuxi, Gonen, Shane, Gonen, Tamir, & Yeates, Todd O. Near-atomic cryo-EM imaging of a small protein displayed on a designed scaffolding system. United States. doi:10.1073/pnas.1718825115.
Liu, Yuxi, Gonen, Shane, Gonen, Tamir, and Yeates, Todd O. Mon . "Near-atomic cryo-EM imaging of a small protein displayed on a designed scaffolding system". United States. doi:10.1073/pnas.1718825115.
@article{osti_1423732,
title = {Near-atomic cryo-EM imaging of a small protein displayed on a designed scaffolding system},
author = {Liu, Yuxi and Gonen, Shane and Gonen, Tamir and Yeates, Todd O.},
abstractNote = {Current single-particle cryo-electron microscopy (cryo-EM) techniques can produce images of large protein assemblies and macromolecular complexes at atomic level detail without the need for crystal growth. However, proteins of smaller size, typical of those found throughout the cell, are not presently amenable to detailed structural elucidation by cryo-EM. Here we use protein design to create a modular, symmetrical scaffolding system to make protein molecules of typical size suitable for cryo-EM. Using a rigid continuous alpha helical linker, we connect a small 17-kDa protein (DARPin) to a protein subunit that was designed to self-assemble into a cage with cubic symmetry. We show that the resulting construct is amenable to structural analysis by single-particle cryo-EM, allowing us to identify and solve the structure of the attached small protein at near-atomic detail, ranging from 3.5- to 5-Å resolution. The result demonstrates that proteins considerably smaller than the theoretical limit of 50 kDa for cryo-EM can be visualized clearly when arrayed in a rigid fashion on a symmetric designed protein scaffold. Furthermore, because the amino acid sequence of a DARPin can be chosen to confer tight binding to various other protein or nucleic acid molecules, the system provides a future route for imaging diverse macromolecules, potentially broadening the application of cryo-EM to proteins of typical size in the cell.},
doi = {10.1073/pnas.1718825115},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 13,
volume = 115,
place = {United States},
year = {2018},
month = {3}
}

Journal Article:
Free Publicly Available Full Text
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DOI: 10.1073/pnas.1718825115

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Cited by: 7 works
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