Molecular goniometers for single-particle cryo-EM of DNA-binding proteins

Correct reconstruction of macromolecular structure by cryo-electron microscopy relies on accurate determination of the orientation of single-particle images. For small (<100 kDa) DNA-binding proteins, obtaining particle images with sufficiently asymmetric features to correctly guide alignment is challenging. DNA nanotechnology was conceived as a potential tool for building host nanostructures to prescribe the locations and orientations of docked proteins. We used DNA origami to construct molecular goniometers—instruments to precisely orient objects—to dock a DNA-binding protein on a double-helix stage that has user-programmable tilt and rotation angles. Each protein orientation maps to a distinct barcode pattern specifying particle classification and angle assignment. We used goniometers to obtain a 6.5 Å structure of BurrH, an 82-kDa DNA-binding protein whose helical pseudosymmetry prevents accurate image orientation using classical cryo-EM. Our approach should be adaptable for other DNA-binding proteins, and a wide variety of other small proteins, by fusing DNA binding domains to them.