DNA Nanostructures for High-Throughput Cryo-EM Studies of Small Macromolecules Single particle cryo-electron microscopy (cryo-EM) is an approach for visualizing structures of macromolecules and their complexes at near-native conditions without the need for large sample quantities or the removal of flexible regions often required for alternative techniques such as X-ray crystallography. Improvements in microscopy hardware and software have helped to achieve near- atomic structure determination of macromolecules by cryo-EM allowing, for example, the visualization of individual amino-acid side chains of protein targets. However, structure determination by cryo-EM remains challenging for many macromolecules. Small (<100 kDa) particles can be especially difficult to study because they lack well-defined structural features required for the image alignment step of 3D reconstruction. To address these challenges, we constructed tiny goniometers—instruments that precisely orient objects—using DNA nanotechnology. Our molecular goniometers can dock a single DNA-binding protein onto a linear double-helix stage. With the power of DNA origami design, we can make several goniometer variants, each with user-defined tilt and rotation angles of the protein-DNA complex. The goniometers include asymmetric barcode domains. Thus, we can bind and precisely orient small DNA-binding proteins, easily identify them on the grid, correctly assign the angle priors, and rapidly solve their structure. We have validated our approach by obtaining a 6.5-Å structure of an 82-kDa DNA-binding protein whose helical pseudosymmetry prevents accurate image orientation using traditional cryo-EM. We propose to apply our DNA origami molecular goniometers to characterize several biomedically important DNA-binding proteins of unknown structure that remain recalcitrant to cryo-EM analysis. We will study nuclear receptors linked to pancreatic and breast cancer, inflammatory bowel disease, diabetes, and nonalcoholic fatty liver disease. We will also study small transcription factors that regulate pleiotropic drug resistance in yeast. That structural understanding may aid in developing future antifungal therapies. We will build custom molecular goniometers for each protein target with the appropriate DNA-binding site and any necessary structural modifications. We will continue to optimize our first-generation designs to address outstanding challenges related to sample preparation and extend the devices for general use with non-DNA binding proteins. These engineering efforts will be aided by computational modeling and simulation tools that our lab has developed and will adapt for each target system.