推进液体和冰中病毒组件的高分辨率成像
1Department of Biomedical Engineering,Pennsylvania State University, 2Department of Materials Science and Engineering,McMaster University, 3Bioinformatics and Genomics Graduate Program, Huck Institutes of the Life Sciences,Pennsylvania State University, 4Molecular, Cellular, and Integrative Biosciences Graduate Program, Huck Institutes of the Life Sciences,Pennsylvania State University, 5Materials Research Institute,Pennsylvania State University, 6Huck Institutes of the Life Sciences,Pennsylvania State University, 7Applications team,Direct Electron, 8Application Scientist,Protochips, Inc., 9Department of Biology,Wake Forest University
Summary
这里描述了使用透射电子显微镜制备适用于纳米级液体电镜和冷冻电镜分析的病毒组件的协议。
Abstract
近年来，人们对液电子显微镜（liquid-EM）的兴趣激增，因为科学家现在可以观察纳米级的实时过程。将高分辨率冷冻电镜信息与动态观察相结合是非常理想的，因为许多事件发生在快速的时间尺度上 – 在毫秒范围内或更快。提高对柔性结构的了解也有助于设计新型试剂来对抗新出现的病原体，例如SARS-CoV-2。更重要的是，在流体环境中观察生物材料可以对它们在人体中的表现进行独特的一瞥。这里介绍的是新开发的方法，用于研究液体和玻璃冰中病毒组装的纳米级特性。为了实现这一目标，使用定义明确的样本作为模型系统。并排比较了样品制备方法和代表性结构信息。显示了在~3.5-Å-10 Å范围内解析的结构的亚纳米特征。支持这一互补框架的其他最新结果包括候选疫苗和液体成像的基于抗体的疗法的动态见解。总体而言，这些相关应用提高了我们可视化分子动力学的能力，为它们在人类健康和疾病的使用提供了独特的背景。
Introduction
生物医学研究通过开发新技术提高了我们对人类健康和疾病的了解。高分辨率成像正在改变我们对纳米世界的看法 – 使我们能够以精致的细节研究细胞和分子1，2，3，4，5。动态组件（如软聚合物、蛋白质组装或人类病毒）的静态信息仅揭示了其复杂叙述的有限快照。为了更好地了解分子实体如何运作，必须共同研究它们的结构和功能。
原子薄石墨烯或硅基微芯片等材料生产的最新进展为使用透射电子显微镜（TEM）进行实时结构函数分析提供了新的机会。这些材料可以创建用于实时EM成像的密封室6，7，8，9，10，11。液电镜的新领域，室温与冷冻电镜相关，提供了溶液中硬质或软质材料的前所未有的视图，使科学家能够同时研究其样品的结构和动力学。液体电镜应用包括治疗性纳米颗粒与癌症干细胞相互作用的实时记录，以及病毒病原体12，13，14分子复杂性的变化。
正如方法学的进步刺激了冷冻电镜领域的分辨率革命一样，需要新的技术和方法来扩展液电镜作为科学界高通量工具的使用。本文介绍的方法的总体目标是简化液电镜样品制备方案。所开发技术背后的基本原理是采用新的微芯片设计和自动加载器设备，适用于液体和冷冻电镜数据收集（图1）7，14，15，16，17。组件使用自动化仪器的标准网格夹进行机械密封，例如 Krios，每个会话可容纳多个样品或 F200C TEM（图 2）。这种方法将高分辨率成像的使用扩展到标准冷冻电镜应用之外，展示了实时材料分析的更广泛用途。
在当前的视频文章中，介绍了在有或没有市售标本支架的情况下在液体中制备病毒组件的实验方案。使用用于液电镜的专用试样支架，薄液体试样可以提供与冷冻电镜样品相当的结构信息，以及试样的动态见解。还展示了使用自动进样器工具制备液体标本的方法，用于高通量常规。与其他技术相比，自动化样品生产的主要优点是允许用户在数据收集之前快速评估样品的最佳厚度和电子剂量。这种筛选技术可快速识别液体或冰12，14，18，19中实时记录的理想区域。出于 3D 结构测定的目的，液电镜可以补充冷冻电镜中实施的长期建立的冷冻电镜方法。采用传统TEM或冷冻电镜技术的读者可以考虑使用液电镜工作流程，以补充其当前策略的方式提供新的样品动态观察结果。
本协议中使用的病毒样品包括作为礼物获得并在标准条件下培养的纯化的腺相关病毒亚型3（AAV）12。还使用了从COVID-19患者血清中提取的非传染性SARS CoV-2亚病毒组件12，并从商业来源获得。最后，从维克森林大学的Sarah M. McDonald Esstman博士的实验室获得纯化的猿猴轮状病毒（SA11菌株）双层颗粒（DLP），并使用标准条件 6，17进行培养。此处描述的软件包是免费提供的，链接已在 “材料表 ”部分中提供。
Protocol
1. 装载液体电磁的试样支架 通过将每个芯片在 150 mL 丙酮中孵育 2 分钟，然后在 150 mL 甲醇中孵育 2 分钟来清洁氮化硅 （SiN） 微芯片。让芯片在层流气流中干燥。 使用辉光放电仪器使用辉光放电仪器清洁干燥的芯片，使用氩气在 30 W、15 mA 的标准条件下运行 45 秒。 将干燥的基础微芯片装入样品支架的尖端。向基础芯片中加入 ~0.2 μL 样品（在 50 mM HEPES、pH 7.5、1…
Representative Results
所有液电镜成像实验均使用工作在200 kV的液电镜，工作电压为300 kV的冷冻电镜用于所有冷冻电镜数据收集。展示了多种病毒的代表性图像和结构，以证明这些方法在各种测试对象中的实用性。这些包括重组腺相关病毒亚型 3 （AAV）、源自患者血清的 SARS-CoV-2 亚病毒组件以及猿轮状病毒双层颗粒 （DLP）、SA11 菌株。首先，演示在液体缓冲溶液（50 mM HEPES，pH 7.5;150 mM NaCl;10 mM MgCl2;10 mM CaCl2<…
Discussion
通过使用从冷冻电镜领域改编的新的自动化工具和技术，为简化当前的液电镜工作流程提供了新的机会。与其他方法相比，涉及新的微芯片夹层技术的应用具有重要意义，因为它们能够在液体或玻璃体冰中进行高分辨率成像分析。该协议中最关键的步骤之一是生产具有理想液体厚度的标本，以可视化纳米级的精美细节。在实施高通量例程进行自动数据收集之前，通过筛选整个样品，以较低的放大?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢Luk H. Vandenberghe博士（哈佛医学院眼科）提供纯化的AAV-3。这项工作得到了美国国立卫生研究院和国家癌症研究所（R01CA193578，R01CA227261，R01CA219700至DFK）的支持。
Materials
| Acetone | Fisher Scientific | A11-1 | 1 Liter |
| Autoloader clipping tool | ThermoFisher Scientific | N/A | Also SubAngstrom supplier |
| Autoloader grid clips | ThermoFisher Scientific | N/A | top and bottom clips |
| Carbon-coated gold EM grids | Electron Microcopy Sciences | CF400-AU-50 | 400-mesh, 5-nm thickness |
| COVID-19 patient serum | RayBiotech | CoV-Pos-S-500 | 500 microliters of PCR+ serum |
| Methanol | Fisher Scientific | A412-1 | 1 Liter |
| Microwell-integrad microchips | Protochips, Inc. | EPB-42A1-10 | 10×10-mm window arrays |
| TEMWindows microchips | Simpore Inc. | SN100-A10Q33B | 9 large windows, 10-nn thick |
| TEMWindows microchips | Simpore, Inc. | SN100-A05Q33A | 9 small windows, 5-nm thick |
| Top microchips | Protochips, Inc. | EPT-50W | 500 mm x 100 mm window |
| Whatman #1 filter paper | Whatman | 1001 090 | 100 pieces, 90 mm |
| Equipment | |||
| DirectView direct electron detector | Direct Electron | 6-micron pixel spacing | |
| Falcon 3 EC direct electron detector | ThermoFisher Scientific | 14-micron pixel spacing | |
| Gatan 655 Dry pump station | Gatan, Inc. | Pump holder tip to 10-6 range | |
| Mark IV Vitrobot | ThermoFisher Scientific | state-of-the-art specimen preparation unit | |
| PELCO easiGlow, glow discharge unit | Ted Pella, Inc. | Negative polarity mode | |
| Poseidon Select specimen holder | Protochips, Inc. | FEI compatible;specimen holder | |
| Talos F200C TEM | ThermoFisher Scientific | 200 kV; Liquid-TEM | |
| Titan Krios G3 | ThermoFisher Scientific | 300 kV; Cryo-TEM | |
| Freely available software | Website link | Comments (optional) | |
| cryoSPARC | https://cryosparc.com/ | other image processing software | |
| CTFFIND4 | https://grigoriefflab.umassmed.edu/ctffind4 | CTF finding program | |
| MotionCorr2 | https://emcore.ucsf.edu/ucsf-software | ||
| RELION | https://www3.mrc-lmb.cam.ac.uk/relion/index.php?title=Main_Page | ||
| SerialEM | https://bio3d.colorado.edu/SerialEM/ | ||
| UCSF Chimera | https://www.cgl.ucsf.edu/chimera/ | molecular structure analysis software package |
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Cite This Article
DiCecco, L., Berry, S., Jonaid, G. M., Solares, M. J., Kaylor, L., Gray, J. L., Bator, C., Dearnaley, W. J., Spilman, M., Dressel-Dukes, M. J., Grandfield, K., McDonald Esstman, S. M., Kelly, D. F. Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice. J. Vis. Exp. (185), e63856, doi:10.3791/63856 (2022).