Jenkins Matthew C, Bobe Daija, Johnston Jake D, Cheung Jonah, Karasawa Akira, Zimanyi Christina M, Dermanci Ömer, Finn M G, de Marco Alex, Kopylov Mykhailo
School of Chemistry and Biochemistry, School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA, 30332, USA.
Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, 10027, USA.
bioRxiv. 2025 Aug 18:2025.08.18.667046. doi: 10.1101/2025.08.18.667046.
Contact with the air-water interface can bias the orientation of macromolecules during cryo-EM sample preparation, leading to uneven sample distribution, preferred orientation, and damage to the molecules of interest. To prevent this, we describe a method to encapsulate target proteins within highly hydrophilic, structurally homogeneous, and stable protein shells, which we refer to as "nanocrates" for this purpose. Here, we describe packaging, data acquisition, and reconstruction of three proof-of-principle examples, each illuminating a different aspect of the method: apoferritin (ApoF, demonstrating high-resolution), thyroglobulin (Tg, solving a known preferred orientation problem), and 7,8-dihydroneopterin aldolase (DHNA, a structure previously uncharacterized by cryo-EM).
在冷冻电镜样品制备过程中，与气-水界面接触会使大分子的取向产生偏差，导致样品分布不均、择优取向以及对目标分子造成损伤。为防止这种情况发生，我们描述了一种将目标蛋白封装在高度亲水、结构均匀且稳定的蛋白质外壳内的方法，为此我们将这种外壳称为“纳米容器”。在此，我们描述了三个原理验证示例的封装、数据采集和重建过程，每个示例都阐明了该方法的不同方面：脱铁铁蛋白（ApoF，展示高分辨率）、甲状腺球蛋白（Tg，解决已知的择优取向问题）以及7,8-二氢新蝶呤醛缩酶（DHNA，一种此前冷冻电镜未表征过的结构）。
