Name: preparation of high-temperature sample grids for cryo-em
Uploaded: 2021-07-26T15:00:00
Description: Watch this Scientific Journal Video about Preparation of High-Temperature Sample Grids for Cryo-EM at JoVE.com

作者感谢赛默飞世尔科技的Hervé Remigy博士提供的有用建议。冷冻电镜实验在中央研究院冷冻电镜设施（ASCEM）进行。ASCEM由中央研究院支持（批准号）。AS-CFII-108-110）和台湾蛋白质计划（批准号。AS-KPQ-109-TPP2）。作者还感谢黄慧菊女士在样品制备方面的帮助。

注意：本协议旨在使用改进的商业玻璃化设备在特定温度下（特别是高于37°C）制备冷冻电子显微镜（cryo-EM）样品。 整体实验设置如图 1所示。该方案以55°C为例。其他温度下的具体条件请参考参考文献12中的补充表2。
1.玻璃化装置的制备
<ol><li>在封闭端的 50 mL 离心管中打一个 1 cm 的孔。</li>
	<li>将管子放入超声波出水口的玻璃化装?...

<ol>
	<li>Yip, K. M., Fischer, N., Paknia, E., Chari, A., Stark, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic-resolution+protein+structure+determination+by+cryo-EM.">Atomic-resolution protein structure determination by cryo-EM.</a> Nature. 587, 157-161 (2020).</li><li>Nakane, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-particle+cryo-EM+at+atomic+resolution.">Single-particle cryo-EM at atomic resolution.</a> Nature. 587, 152-156 (2020).</li><li>Chen, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Cryo-EM+to+uncover+structural+bases+of+pH+effect+and+cofactor+bi-specificity+of+ketol-acid+reductoisomerase.">Use of Cryo-EM to uncover structural bases of pH effect and cofactor bi-specificity of ketol-acid reductoisomerase.</a> Journal of the American Chemical Society. 141, 6136-6140 (2019).</li><li>Cabra, V., Sams&#243;, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do's+and+don'ts+of+cryo-electron+microscopy:+A+primer+on+sample+preparation+and+high+quality+data+collection+for+macromolecular+3D+reconstruction.">Do's and don'ts of cryo-electron microscopy: A primer on sample preparation and high quality data collection for macromolecular 3D reconstruction.</a> Journal of Visualized Experiments. (95), e52311 (2015).</li><li>Klebl, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Need+for+speed:+Examining+protein+behavior+during+CryoEM+grid+preparation+at+different+timescales.">Need for speed: Examining protein behavior during CryoEM grid preparation at different timescales.</a> Structure. 28 (11), 1238-1248 (2020).</li><li>Passmore, L. A., Russo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specimen+preparation+for+high+resolution+cryo-EM.">Specimen preparation for high resolution cryo-EM.</a> J. Methods in Enzymology. 579, 51-86 (2016).</li><li>Laughlin, T. G., Bayne, A. N., Trempe, J. -. F., Savage, D. F., Davies, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+complex+I-like+molecule+NDH+of+oxygenic+photosynthesis.">Structure of the complex I-like molecule NDH of oxygenic photosynthesis.</a> Nature. 566, 411-414 (2019).</li><li>Gao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures+and+operating+principles+of+the+replisome.">Structures and operating principles of the replisome.</a> Science. 363, (2019).</li><li>Zhao, Y., Chen, S., Swensen, A. C., Qian, W. -. J., Gouaux, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+and+subunit+arrangement+of+native+AMPA+receptors+elucidated+by+cryo-EM.">Architecture and subunit arrangement of native AMPA receptors elucidated by cryo-EM.</a> Science. 364, 355-362 (2019).</li><li>Chen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+dynamics+of+ribosome+subunit+association+studied+by+mixing-spraying+time-resolved+cryogenic+electron+microscopy.">Structural dynamics of ribosome subunit association studied by mixing-spraying time-resolved cryogenic electron microscopy.</a> Structure. 23, 1097-1105 (2015).</li><li>Singh, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+temperature+sensation+by+the+TRP+channel+TRPV3.">Structural basis of temperature sensation by the TRP channel TRPV3.</a> Nature Structure and Molecular Biology. 26, 994-998 (2019).</li><li>Chen, C. Y., Chang, Y. C., Lin, B. L., Huang, C. H., Tsai, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-resolved+cryo-EM+uncovers+structural+bases+of+temperature-dependent+enzyme+functions.">Temperature-resolved cryo-EM uncovers structural bases of temperature-dependent enzyme functions.</a> Journal of the American Chemical Society. 141, 19983-19987 (2019).</li></ol>

在协议的步骤1中，确保离心管安装良好，并且在实验进行时不会掉落。由于腔室内积聚了大量的水滴，会改变滤纸的吸附能力，建议玻璃化装置腔达到平衡温度后，实验的总时间不应超过30分钟。如果操作时间超过30分钟，操作人员需要更换滤纸，等待机舱再次平衡温度和湿度。在协议的第8步，建议的7-9μL样品体积比平时大，因为否则，样品在高温下会迅速蒸发，导致网格上出现空方块。另一方?...

用于求解蛋白质复合物结构的冷冻电镜技术不断改进，特别是在获得高分辨率结构的方向上1，2.同时，在玻璃化过程3之前，通过改变样品条件（例如pH或配体）也扩大了其应用范围，该过程涉及制备样品网格，然后进行冷冻4，5。另一个重要条件是温度。尽管冷冻电镜实验（如X射线晶体学）是在低温下进行的，但冷冻电镜求解的结构反映了玻璃化前溶液状态下的结构。直到最近，大多数单颗粒分析 （SPA） 冷冻电镜研究使用在玻璃化之前保存在冰上的样品6，尽管许多研究使用室温约为 7，8，9，10 或高达 42 °C的样品 11。在最近的一份报告中，我们对嗜热古菌Sulfolobus solfataricus（Sso）的酮酸还原异构酶（KARI）在4°C至70°C的六种不同温度下进行了温度依赖性研究12。我们的研究表明，在功能相关的温度下制备样品网格非常重要，冷冻电镜是唯一在多个温度下求解相同蛋白质复合物结构的实际可行的结构方法。
高温玻璃化的主要难点是尽量减少蒸汽冷凝并实现薄冰。在这里，我们报告了我们之前对 Sso-KARI 12的研究中用于在高温下制备样品网格的详细方案。我们假设读者或观众已经对冷冻电镜实验的整体样品制备和数据处理程序有经验，并强调与高温相关的方面。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Falcon tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td>size: 50 mL</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Ted Pella</td>
			<td>47000-100</td>
			<td>&#216;55/20mm, Grade 595</td>
		</tr>
		<tr>
			<td>HI1210</td>
			<td>Leica</td>
			<td></td>
			<td>water bath</td>
		</tr>
		<tr>
			<td>K100X</td>
			<td>Electron Microscopy Sciences</td>
			<td></td>
			<td>glow discharge</td>
		</tr>
		<tr>
			<td>Quantifoil, 1.2/1.3 200Mesh Cu grid</td>
			<td>Ted Pella</td>
			<td>658-200-CU-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Titan Krios G3</td>
			<td>Thermo Fisher Scientific</td>
			<td>1063996</td>
			<td>low dose imaging</td>
		</tr>
		<tr>
			<td>Vitrobot Mark IV</td>
			<td>Thermo Fisher Scientific</td>
			<td>1086439</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitrobot Tweezer</td>
			<td>Ted Pella</td>
			<td>47000-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of high-temperature sample grids for cryo-em

用于冷冻电子显微镜（cryo-EM）实验的样品网格通常在最适合生物样品储存的温度下制备，主要是在4°C下，偶尔在室温下制备。最近，我们发现在低温下解决的蛋白质结构可能与功能无关，特别是对于嗜热古细菌的蛋白质。开发了一种在较高温度（高达70°C）下制备蛋白质样品以进行冷冻电镜分析的程序。我们发现，在较高温度下制备的样品的结构具有功能相关性且与温度相关。在这里，我们以55°C为例描述了在高温下制备样品网格的详细方案。该实验使用使用额外的离心管修饰的玻璃化装置，并将样品在55°C下孵育。 对详细程序进行了微调，以最大程度地减少蒸汽冷凝并在网格上获得一层薄薄的冰。提供了成功和不成功的实验示例。

低放大倍率概览如图 5A，B所示。面板 A 是成功网格的一个示例。从左上角（较厚）到右下角（较薄或空）有一个冰梯度。这样的网格可以更容易地在适合数据收集的中间区域找到合适厚度的冰层，例如蓝色和绿色框。网格 B 太干。网格中的方块具有明亮的对比度，这意味着冰层太薄或根本没有冰层。只有红色箭头指示的两个方块适合数据收集。
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Watch this Scientific Journal Video about Preparation of High-Temperature Sample Grids for Cryo-EM at JoVE.com

Preparation of High-Temperature Sample Grids for Cryo-EM

本文提供了在冷冻电镜实验进行冷冻之前在高达 70 °C 的温度下制备样品网格的详细方案。

冷冻电镜高温样品网格的制备

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, ...

蛋白质的功能是温度依赖性的，并且在温度下是最佳的。该协议提供了一种在较高温度下解决蛋白质结构的方法。这种方法可以提供功能相关的结构，并增强对蛋白质结构温度依赖性的理解。演示该程序的将是中央研究院冷冻电镜设施经理张元志。首先在其封闭端的 50 毫米离心管上打一个 1 厘米的孔。将管子放在超声波出水口的玻璃化装置室中。将玻璃化装置温度设置为指定温度，并允许玻璃化装置室达到55摄氏度和100%相对湿度。在开始实验之前，让它静置至少半小时以稳定条件。将水浴放在热板上，并将热板设置为所需温度。用温度计检查以确保水达到55摄氏度。在水浴中孵育样品，并在印迹实验之前将移液器吸头在热板边缘预热两分钟或更长时间。辉光以 25 毫安的电流将多孔碳支撑的网格放电 30 秒。在印迹实验前不早于五分钟将玻璃化滤纸放入玻璃化装置室中。将镊子与玻璃化装置中的网格一起孵育两分钟或更长时间。按照标准程序用乙烷构建乙烷容器，确保乙烷不会溢出。使用移液器将 7 到 9 微升的样品涂在网格上。然后等待一到两秒钟，吸干一到一秒半，然后迅速将样品浸入液态乙烷中。将网格从液态乙烷转移到储存在液氮中的冷冻盒中。夹住网格并卸载它们以获取电子显微镜或冷冻电镜仪器。使用冷冻电镜仪器的显示屏和软件的低剂量功能，在网格上筛选冰况和样品在网格上的分布。如果所得网格的质量不好，请在等待时间、印迹时间等各种条件下重复网格准备过程。如果生成的网格质量良好，请重复相同的步骤以在不同的温度下制作网格。将高质量的网格转移到高分辨率冷冻电镜仪器上。根据既定程序执行数据收集和数据分析。在成功网格的情况下，形成冰梯度，网格左上角的冰较厚，右下角的冰较薄。在成功的网格中观察到适合数据收集的蓝色和绿色框。在另一个网格中观察到了明亮的对比。表示薄薄的一层，冰或没有冰。在第二个网格中，只有两个方块适合数据收集。其中一个网格由冰组成，主要以晶体形式组成，不适合数据收集。另一方面，另一个网格显示冰层大多处于适合数据收集的无定形状态。作为容器和其他容器的准备工作需要根据时间点完成。时间控制和快速准确地完成实验是最重要的。根据使用该协议获得的结果，研究人员在设置简单条件时可能必须更加小心。

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