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

biorxiv. 2020; 
Tural Aksel,  Zanlin Yu,   Yifan Cheng,  Shawn M. Douglas
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Molecular Biology Reagents The BurrH open reading frame (ORF) with a flexible linker and C-terminal HIS TAG (-GSGHHHHHH) was subcloned into pet24d+ expression vector (Genscript, NJ, USA). Get A Quote

摘要

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 pa... More

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