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

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 protein 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 muscle aldolase through a helical linker. The DARPin retained its ability to bind its target, the 27 kDa green fluorescent protein (GFP). We solved 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 describe strategies to rigidify this element. Author summary Single particle cryogenic electron microscopy (cryoEM) is a technique that uses images of purified proteins to determine their atomic structure. Unfortunately, the majority of proteins in the human and bacterial proteomes are too small to be analyzed by cryoEM. Over the years, several groups have suggested the use of a platform to increase the size of small protein targets. The platform is composed of a large protein base and a selectable adapter that binds the target protein. Here we report a platform based on tetrameric rabbit muscle aldolase that is fused to a Designed Ankyrin Repeat Protein (DARPin). Phage display libraries can be used to generate DARPins against target proteins. The residues mutated in a phage display library to generate a DARPin against a new target do not overlap with the DARPin-base fusion in the platform, thus changing the DARPin identity will not disrupt the platform design. The DARPin adapter used here is capable of binding Green Fluorescent Protein (GFP). We report the structure of GFP to 5 to 8 Å local resolution by single particle cryoEM. Our analysis demonstrates that flexibility in the DARPin-aldolase platform prevents us from achieving higher resolution in the GFP region. We suggest changes to the DARPin design to rigidify the DARPin-aldolase platform. This work expands on current platforms and paves a generally applicable way toward structure determination of small proteins by cryoEM.