小分子的微晶电子衍射
Summary
在这里，我们描述了我们实验室开发的用于制备用于微晶电子衍射（MicroED）实验的小分子晶体粉末的程序。
Abstract
描述了制备用于微晶电子衍射（MicroED）实验的小分子样品的详细方案。MicroED已被开发用于使用标准电子冷冻显微镜（cryo-EM）设备解决蛋白质和小分子的结构。通过这种方式，小分子、肽、可溶性蛋白和膜蛋白最近被测定到高分辨率。这里介绍了以药物卡马西平为例制备小分子药物网格的方案。提出了筛选和收集数据的协议。整个过程中的其他步骤（如数据集成、结构确定和优化）将在别处介绍。准备小分子网格所需的时间估计不到30分钟。
Introduction
微晶电子衍射（MicroED）是一种电子冷冻显微镜（cryo-EM）方法，用于从亚微米尺寸的晶体1，2中确定原子分辨率结构。将晶体应用于标准透射电子显微镜（TEM）网格，并通过投入液态乙烷或液氮进行冷冻。然后将网格加载到在低温下运行的TEM中。晶体位于网格上并筛选初始衍射质量。连续旋转 MicroED数据是从筛选晶体的子集中收集的，其中数据使用快速相机保存为电影3。这些电影被转换为标准的晶体学格式，并且处理方式几乎与X射线晶体学实验4相同。
MicroED最初是为了研究蛋白质微晶1，2而开发的。蛋白质晶体学的一个瓶颈是为传统的同步加速器X射线衍射实验生长出大而有序的晶体。由于电子与比X射线强几个数量级的物质相互作用，产生可检测衍射所需的晶体尺寸的限制要小得多5。此外，弹性与非弹性散射事件的比例对电子更有利，这表明可以在较小的整体暴露5下收集更多有用的数据。不断发展使得MicroED数据可以从最具挑战性的微晶6，7，8，9中收集。
最近，MicroED已被证明是从明显无定形的材料10，11，12，13确定小分子药物结构的有力工具。这些粉末可以直接来自一瓶购买的试剂、纯化柱，甚至可以来自将药丸粉碎成细粉10。这些粉末肉眼看起来是无定形的，但可以完全由纳米晶体组成，或者仅以更大的非结晶、无定形部分含有微量的纳米晶体沉积物。将材料应用于网格是容易的，晶体鉴定、筛选和数据收集的后续步骤甚至可能在不久的将来实现自动化14。虽然其他人可能使用不同的方法来制备样品和数据收集，但这里详细介绍了 Gonen 实验室开发和使用的用于制备 MicroED 的小分子样品和数据收集的协议。
Protocol
1. 制备小分子样品 将少量（0.01 – 1 mg）粉末、液体或固体转移到小瓶或管中。 对于已经呈粉末形式的样品，使用盖子密封试管，直到需要样品为止。在尝试方法1（步骤3）或步骤2（步骤4）之前，将液体样品干燥成粉末。注意：溶解在液体中的样品可以使用下面的方法3（5.X） 2. 准备透射电镜网格 注意：一些带有自动加载系统的TEM要?…
Representative Results
MicroED是一种冷冻电镜方法，它利用电子和物质之间的强相互作用，可以研究消失的小晶体12，13。在这些步骤之后，有望从微晶中收集晶体学格式的衍射电影（视频1）。在这里，使用卡马西平12演示该技术。结果显示了来自在TEM网格上鉴定的卡马西平微晶的连续旋转MicroED数据集（视频1）。一个好的数据集具有…
Discussion
样品制备通常是一个迭代过程，在筛选和数据收集后进行优化。对于小分子样品，通常谨慎的做法是首先尝试网格制备而不对网格进行辉光放电，因为许多药物往往是疏水性的10，11。如果网格的纳米晶沉积物太少，最好在第一次对网格进行辉光放电后重试。可能是冻干粉末的晶体太大太厚，无法收集良好的数据。在这些情况下，可以从较大晶体的边缘?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Gonen实验室得到了霍华德休斯医学研究所的资金支持。这项研究得到了美国国立卫生研究院P41GM136508的支持。
Materials
| 0.1-1.5mL Eppendorf tubes | Fisher Scientific | 14-282-300 | Any vial or tube will do. |
| Autogrid clips | Thermo-Fisher | 1036173 | Clipped grids are not required for MicroED. They are required for Thermo-Fisher TEMs equipped with an autoloader system. |
| Autogrid C-rings | Thermo-Fisher | 1036171 | |
| Carbamazapine | Sigma | C4024-1G | Any amount will suffice for these experiments |
| CMOS based detector | Thermo-Fisher | CetaD 16M | We used a CetaD 16M, but any detector with rolling shutter mode or sufficiently fast readout is acceptable. |
| Delphi software | Thermo-Fisher | N/A | Software on Thermo-Fisher TEM systems that allows for manual rotation of the sample stage |
| EPU-D software | Thermo-Fisher | N/A | Commercial software for the acquisition of MicroED data |
| Glass cover slides | Hampton | HR3-231 | |
| Glow discharger | Pelco | easiGlow | |
| High PrecisionTweezers | EMS | 78325-AC | Any high precision tweezer will do |
| Liquid nitrogen vessel | Spear Lab | FD-800 | A standard foam vessel for handling specimens under liquid nitrogen – 800mL |
| SerialEM software | UC Boulder | N/A | Free software distributed by D. Mastronarde. Department of Molecular, Cellular, and Developmental Biology |
| TEM grids | Quantifoil/EMS | Q310CMA | Multi-A 300 mesh grids were used here, but any thin carbon grids will work. For these small molecules, we suggest starting with continuous carbon. |
| transmission electron microscope (TEM) | Thermo-Fisher | Talos Arctica | |
| Whatman circular filter paper | Millipore-Sigma | WHA1001090 | 90mm or larger |
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Cite This Article
Martynowycz, M. W., Gonen, T. Microcrystal Electron Diffraction of Small Molecules. J. Vis. Exp. (169), e62313, doi:10.3791/62313 (2021).