Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA.
Howard Hughes Medical Institute and the Department of Biochemistry & Biophysics, University of California, San Francisco, San Francisco, CA, USA.
Nat Biotechnol. 2021 Mar;39(3):378-386. doi: 10.1038/s41587-020-0716-8. Epub 2020 Oct 19.
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-Å 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.
通过冷冻电子显微镜（cryo-EM）正确重建大分子结构依赖于准确确定单粒子图像的方向。对于小的（<100 kDa）DNA 结合蛋白，获得具有足够非对称特征以正确指导对准的粒子图像是具有挑战性的。我们应用 DNA 折纸术来构建分子测角器-精确定向物体的仪器-并将其用于将 DNA 结合蛋白固定在具有用户可编程倾斜和旋转角度的双螺旋台上。我们构建了具有 14 种不同台配置的测角器，以在 cryo-EM 网格表面上方定位和可视化蛋白质。每个测角器都有一个独特的条码模式，我们在粒子分类过程中使用该模式将角度先验分配给结合的蛋白质。我们使用测角器获得了 BurrH 的 6.5-Å 结构，BurrH 是一种 82 kDa 的 DNA 结合蛋白，其螺旋拟对称性阻止了使用传统 cryo-EM 进行准确的图像定向。我们的方法应该适应于其他 DNA 结合蛋白以及与 DNA 结合域融合的小蛋白。
