Mar 7, 2018

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

Proceedings of the National Academy of Sciences of the United States of America
Yuxi LiuT O Yeates

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 ...Continue Reading

  • References42
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References

  • References42
  • Citations3

Citations

Mentioned in this Paper

Signaling Molecule
Muscle Rigidity
Macromolecular Compounds
Size
Complex (molecular entity)
Gene Products, Protein
Crystallography, X-Ray
Scaffold protein
Staphylococcal Protein A
Macromolecular Complexes

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