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

Abstract

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.

Advances in methods for atomic resolution macromolecular structure determination

“…Specific loop sequence mutations in the DARPin (or other adaptor protein) required to bind a given cargo protein can be identified experimentally based on separate laboratory evolution studies (see 65 for a recent review of DAPRin applications). Sub-4 Å resolution has been achieved by a scaffolding system of this type 57 . An important advance in visualizing RNA molecules in the 40 kDa range has also been demonstrated recently using a Volta phase plate to enhance image contrast, resulting in sub-4 Å resolution 66 .…”

Synthesis and Evaluation of 4-Hydroxycoumarin Imines as Inhibitors of Class II Myosins

“…Three 45-60 s movies were collected per slide with three slides per inhibitor concentration. The movies were analyzed with FAST software 64,65 using a 20% filtering cutoff and five-frame minimum. The filaments shorter than 1 μm were excluded.…”

Modular Protein–DNA Cocrystals as Precise, Programmable Assembly Scaffolds

“…Scaffold-assisted X-ray crystallography is a potentially high-throughput structural biology strategy that decouples the crystallization problem from the target biomolecule, reviewed previously. 16 Limited examples of scaffolds using DNA-binding for host–guest structural biology include cryo-EM goniometers 17 and protein–DNA host crystals for small-molecule drugs. 18 Here, we pursue a scaffold lattice that can be optimized independently of the guest.…”