单层石墨烯在冷冻电子显微镜网格中的应用，用于高分辨率结构测定
Summary
在低温电子显微镜（cryoEM）网格中应用支撑层可以增加颗粒密度，限制与空气-水界面的相互作用，减少光束引起的运动，并改善颗粒取向的分布。本文描述了一种用单层石墨烯涂覆冷冻电镜网格以改进冷冻样品制备的稳健方案。
Abstract
在低温电子显微镜（cryoEM）中，纯化的大分子被施加到带有多孔碳箔的网格上;然后将分子吸干以除去多余的液体，并迅速冻结在大约20-100nm厚的玻璃冰层中，悬浮在大约1μm宽的箔孔上。使用低温透射电子显微镜对所得样品进行成像，并使用合适的软件进行图像处理后，可以确定近原子分辨率的结构。尽管冷冻电镜已被广泛采用，但样品制备仍然是冷冻电镜工作流程中的一个严重瓶颈，用户经常遇到与样品在悬浮玻璃冰中表现不佳相关的挑战。最近，已经开发出用单个连续石墨烯层修改冷冻电镜网格的方法，石墨烯作为支撑表面，通常增加成像区域中的颗粒密度，并可以减少颗粒与空气-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冷冻电镜网格的详细方案，并用于快速评估所得网格的相对亲水性。此外，我们还描述了一种基于电磁的方法，通过可视化石墨烯的特征衍射图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2.7 Å分辨率密度图，证明了这些石墨烯载体的实用性。
Introduction
单颗粒低温电子显微镜 （cryoEM） 已发展成为一种广泛使用的可视化生物大分子的方法1。在直接电子检测2、3、4、数据采集5 和图像处理算法6、7、8、9、10 的进步推动下，cryoEM 现在能够产生快速增长的大分子数量的近原子分辨率3D 结构 11.此外，通过利用该方法的单分子性质，用户可以从单个样品12,13,14,15中确定多个结构，突出了使用生成的数据来理解异质结构集合16,17的前景。尽管取得了这些进展，但冷冻样本网格制备的瓶颈仍然存在。
为了通过冷冻电镜进行结构表征，生物样品应充分分散在水溶液中，然后必须通过称为玻璃化的过程快速冷冻18,19。目标是捕获悬浮在规则间隔孔上的均匀薄玻璃化冰层中的颗粒，这些孔通常被切割成一层无定形碳。这种图案化的无定形碳箔由带有铜或金支撑棒网的 TEM 网格支撑。在标准工作流程中，在应用样品之前，使用辉光放电等离子体处理使网格具有亲水性。用滤纸吸干多余的液体，使蛋白质溶液在孔上形成一层薄薄的液体膜，在浸入冷冻过程中可以很容易地玻璃化。常见的挑战包括颗粒定位到空气-水界面（AWI）和随后的变性20,21,22或采用优选取向23,24,25，颗粒粘附在碳箔上而不是迁移到孔中，以及孔内颗粒的聚集和聚集26.冰层厚度不均匀是另一个问题;由于电子散射增加，厚冰会导致显微照片中更高水平的背景噪声，而极薄的冰可以排除较大的颗粒27。
为了应对这些挑战，各种薄支撑膜已被用于涂覆网格表面，使颗粒停留在这些支撑物上，理想情况下，避免与空气-水界面的相互作用。石墨烯载体已经显示出巨大的前景，部分原因是它们的高机械强度加上它们最小的散射截面，这减少了支撑层28添加的背景信号。除了对背景噪声的贡献最小外，石墨烯还表现出显着的导电性和导热性29。石墨烯和氧化石墨烯涂层网格已被证明可以产生更高的颗粒密度、更均匀的颗粒分布30，并减少对 AWI22 的定位。此外，石墨烯提供了一个支撑面，可以进一步修改为：1）通过功能化31,32,33调节网格表面的物理化学性质;或 2） 偶联连接剂，促进目标蛋白的亲和纯化 34,35,36。
在本文中，我们修改了用单个均匀的石墨烯30 层涂覆冷冻电镜网格的现有程序。这些修改旨在最大限度地减少整个协议中的网格处理，以提高产量和可重复性。此外，我们还讨论了评估各种紫外线/臭氧处理在使网格在暴跌前具有亲水性的功效的方法。使用石墨烯涂层网格进行冷冻电镜样品制备的这一步骤至关重要，我们发现我们量化所得网格的相对亲水性的简单方法很有用。使用该方案，我们通过与向导RNA和靶DNA复合物生成催化失活的 化脓性 链球菌Cas9的高分辨率3D重建，证明了使用石墨烯包被网格进行结构测定的效用。
Protocol
Representative Results
Discussion
冷冻电镜样品制备涉及许多技术挑战，大多数工作流程要求研究人员极其小心地手动操作易碎的网格，以避免损坏它们。此外，任何样品对玻璃化的适应性都是不可预测的;颗粒经常与空气-水界面或覆盖网格的固体支撑箔相互作用，这可能导致颗粒采用首选方向或无法进入成像孔，除非施加非常高的蛋白质浓度24。用连续的单层石墨烯覆盖多孔的冷冻电镜网格在改善显微照片上的…
Declarações
The authors have nothing to disclose.
Acknowledgements
标本在麻省理工学院的 CryoEM 设施中制备并成像，该设施使用由 Arnold 和 Mabel Beckman 基金会获得的显微镜。接触角成像设备在麻省理工学院大都会创客空间打印。我们感谢 Nieng Yan 和 Yimo Han 的实验室，以及 MIT.nano 的工作人员在整个采用这种方法的过程中给予的支持。我们特别感谢高冠辉博士和Sarah Sterling博士的深刻讨论和反馈。这项工作得到了 NIH 资助 R01-GM144542、5T32-GM007287 和 NSF-CAREER 资助2046778的支持。戴维斯实验室的研究得到了Alfred P. Sloan基金会，James H. Ferry基金会，麻省理工学院J-Clinic和Whitehead家族的支持。
Materials
| 250 mL beaker (3x) | Fisher | 02-555-25B | |
| 50 mL beaker (2x) | Corning | 1000-50 | |
| Acetone | Fisher | A949-4 | |
| Aluminum foil | Fisher | 15-078-292 | |
| Ammonium persulfate | Fisher | (I17874 | |
| Coverslips 50 mm x 24 mm | Mattek | PCS-1.5-5024 | |
| CVD graphene | Graphene Supermarket | CVD-Cu-2×2 | |
| easiGlow discharger | Ted-Pella | 91000S | |
| Ethanol | Millipore-Sigma | 1.11727 | |
| Flat-tip tweezers | Fisher | 50-239-60 | |
| Glass cutter | Grainger | 21UE26 | |
| Glass petri plate and cover | VWR | 75845-544 | |
| Glass serological pipette | Fisher | 13-676-34D | |
| Grid Storage Case | EMS | 71146-02 | |
| Hot plate | Fisher | 07-770-108 | |
| Isopropanol | Sigma | W292907 | |
| Kimwipe | Fisher | 06-666 | |
| Lab scissors | Fisher | 13-806-2 | |
| Methyl-Methacrylate EL-6 | Kayaku | MMA M310006 0500L1GL | |
| Molecular grade water | Corning | 46-000-CM | |
| Negative action tweezers (2x) | Fisher | 50-242-78 | |
| P20 pipette | Rainin | 17014392 | |
| P200 pipette | Rainin | 17008652 | |
| Parafilm | Fisher | 13-374-12 | |
| Pipette tips | Rainin | 30389291 | |
| Quantifoil grids with holey carbon | EMS | Q2100CR1 | |
| Spin coater | SetCas | KW-4A | with chuck SCA-19-23 |
| Straightedge | ULINE | H-6560 | |
| Thermometer | Grainger | 3LRD1 | |
| UV/Ozone cleaner | BioForce | SKU: PC440 | |
| Vacuum desiccator | Thomas Scientific | 1159X11 | |
| Whatman paper | VWR | 28297-216 |
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