单层石墨烯在用于高分辨率结构测定的冷冻电子显微镜栅格中的应用。

Application of Monolayer Graphene to Cryo-Electron Microscopy Grids for High-resolution Structure Determination.

机构信息

Department of Biology, Massachusetts Institute of Technology.

Department of Biology, Massachusetts Institute of Technology; Program in Computational and Systems Biology, Massachusetts Institute of Technology;

出版信息

J Vis Exp. 2023 Nov 10(201). doi: 10.3791/66023.

Abstract

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to remove excess liquid and rapidly frozen in a roughly 20-100 nm thick layer of vitreous ice, suspended across roughly 1 µm wide foil holes. The resulting sample is imaged using cryogenic transmission electron microscopy, and after image processing using suitable software, near-atomic resolution structures can be determined. Despite cryoEM's widespread adoption, sample preparation remains a severe bottleneck in cryoEM workflows, with users often encountering challenges related to samples behaving poorly in the suspended vitreous ice. Recently, methods have been developed to modify cryoEM grids with a single continuous layer of graphene, which acts as a support surface that often increases particle density in the imaged area and can reduce interactions between particles and the air-water interface. Here, we provide detailed protocols for the application of graphene to cryoEM grids and for rapidly assessing the relative hydrophilicity of the resulting grids. Additionally, we describe an EM-based method to confirm the presence of graphene by visualizing its characteristic diffraction pattern. Finally, we demonstrate the utility of these graphene supports by rapidly reconstructing a 2.7 Å resolution density map of a Cas9 complex using a pure sample at a relatively low concentration.

摘要

在低温电子显微镜(cryoEM)中,将纯化的大分子应用于带有孔状碳箔的网格上;然后将分子吸干以去除多余的液体,并在大约 20-100nm 厚的玻璃态冰层中快速冷冻,该冰层悬浮在大约 1µm 宽的箔孔上。使用低温透射电子显微镜对得到的样品进行成像,然后使用合适的软件进行图像处理,就可以确定接近原子分辨率的结构。尽管 cryoEM 已被广泛采用,但样品制备仍然是 cryoEM 工作流程中的一个严重瓶颈,用户经常遇到与悬浮玻璃态冰中样品表现不佳相关的挑战。最近,已经开发出了用单层石墨烯修饰 cryoEM 网格的方法,石墨烯作为支撑表面,通常可以增加成像区域中的颗粒密度,并减少颗粒与气-水界面之间的相互作用。在这里,我们提供了将石墨烯应用于 cryoEM 网格的详细方案,并介绍了一种快速评估所得网格相对亲水性的方法。此外,我们描述了一种基于 EM 的方法,可以通过可视化其特征衍射图案来确认石墨烯的存在。最后,我们通过使用相对低浓度的纯样品快速重建 Cas9 复合物的 2.7Å 分辨率密度图,证明了这些石墨烯支撑物的实用性。

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