期刊
NATURE BIOTECHNOLOGY
卷 39, 期 3, 页码 378-386出版社
NATURE PORTFOLIO
DOI: 10.1038/s41587-020-0716-8
关键词
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资金
- Ruth L. Kirschstein NRSA Postdoctoral Fellowship grant [F32GM119322]
- UCSF Program for Breakthrough Biomedical Research, Pew-Stewart Scholars Program
- NSF CAREER award [1453847]
- NIH [R35GM125027, R01GM098672, R01HL134183, S10OD021741, S10OD020054]
- Direct For Computer & Info Scie & Enginr [1453847] Funding Source: National Science Foundation
- Division of Computing and Communication Foundations [1453847] Funding Source: National Science Foundation
The researchers used DNA origami technology to create molecular goniometers to assist in accurately positioning small DNA-binding proteins in cryo-electron microscopy for visualization and structural analysis. This approach is particularly effective for DNA-binding proteins with helical pseudosymmetry.
Correct reconstruction of macromolecular structure by cryo-electron microscopy (cryo-EM) 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. We apply DNA origami to construct molecular goniometers-instruments that precisely orient objects-and use them to dock a DNA-binding protein on a double-helix stage that has user-programmable tilt and rotation angles. We construct goniometers with 14 different stage configurations to orient and visualize the protein just above the cryo-EM grid surface. Each goniometer has a distinct barcode pattern that we use during particle classification to assign angle priors to the bound protein. We use goniometers to obtain a 6.5-angstrom structure of BurrH, an 82-kDa DNA-binding protein whose helical pseudosymmetry prevents accurate image orientation using traditional cryo-EM. Our approach should be adaptable to other DNA-binding proteins as well as small proteins fused to DNA-binding domains.
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