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对单粒子图像方向的准确确定。对于小的（<100 kDa）DNA结合蛋白，要获得具有足够不对称特征以正确引导对齐的粒子图像是具有挑战性的。我们使用DNA折纸来构造分子测角仪（一种精确定位对象的仪器），并使用它们将DNA结合蛋白固定在具有用户可编程的倾斜角度和旋转角度的双螺旋平台上。我们构建了具有14种不同阶段配置的测角仪，以定位和可视化cryo-EM网格表面上方的蛋白质。每个测角计都有一个独特的条形码模式，我们在颗粒分类过程中使用该模式来为结合的蛋白质分配先验角度。我们使用测角计获得6。BurrH的5Å结构是82-kDa的DNA结合蛋白，其螺旋假对称性阻止了使用传统cryo-EM的精确图像定向。我们的方法应适用于其他DNA结合蛋白以及与DNA结合域融合的小蛋白。