( 14), specifically those built from 24 subunits, four trimers of two different subunit types. To design a modular cryo-EM scaffold, we took as a starting point a set of protein cages designed by King et al. ( B) Detailed view of the specific scaffold, DARP14, that was designed and characterized in this study, with subunits colored as in A. In principle, binding a macromolecule of interest to the designed scaffold results in the symmetric display of 12 copies of the molecule. The DARPin subunit contains variable loops (highlighted in pink) whose amino acid sequence can be selected to confer binding to a wide range of specific macromolecules of interest. The cryo-EM structure of a binding scaffold is solved in this study. The α-helical termini of the cage subunit (yellow) and the DARPin subunit (red) can be joined in a rigid fashion through genetic fusion, forming a general binding scaffold. At least one of the subunits needs to have an α-helical terminus (cylinder). The example shown is a tetrahedrally symmetric cage with 24 subunits in a 12b 12 stoichiometry (A subunits in yellow and B subunits in blue). ( A) Schematic diagram for a scaffolding system built on a designed symmetric protein cage. A molecular scaffolding system for modular display of macromolecules for cryo-EM imaging. This critical size limitation represents a singular impediment to the universal application of EM for elucidating the structures of most proteins in the human genome.įig. At the other end of the spectrum, however, individual protein molecules of typical size (e.g., 50 kDa or smaller), which lack the aforementioned advantages, remain extremely difficult to visualize at atomic detail by EM. For those reasons, viral capsids are quintessential examples for favorable cryo-EM reconstruction. In such studies, very large macromolecular assemblies offer important advantages in signal processing and imaging, and this advantage is enhanced in systems that are highly symmetric-e.g., composed of numerous repeating copies of one or a few protein building blocks. In favorable cases, 3D cryo-EM image reconstruction methods can produce structures of macromolecular complexes at atomic level detail ( 4– 9). Recent advancements have brought single-particle electron microscopy (EM) techniques to the forefront of structural biology ( 1– 3). 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. 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. 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. 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. Here we use protein design to create a modular, symmetrical scaffolding system to make protein molecules of typical size suitable for cryo-EM. However, proteins of smaller size, typical of those found throughout the cell, are not presently amenable to detailed structural elucidation by cryo-EM. 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.
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