Name: single-particle cryo-em data collection with stage tilt using leginon
Uploaded: 2022-07-01T13:00:00
Description: Watch this Scientific Journal Video about Single-Particle Cryo-EM Data Collection with Stage Tilt using Leginon at JoVE.com

我们感谢Bill Anderson，Charles Bowman和Jean-Christophe Ducom（TSRI）在显微镜，Leginon安装和数据传输基础设施方面的帮助。我们还要感谢Gordon Louie（Salk Institute）和Yong Zi Tan（新加坡国立大学）对手稿的批判性阅读。我们感谢Chris Russo（剑桥MRC分子生物学实验室）为我们提供表达DPS的质粒。这项工作得到了美国国立卫生研究院（U54AI150472，U54 AI170855和R01AI136680到DL），国家科学基金会（NSF MCB-2048095到DL），赫斯特基金会（到DL）和Arthur和Julie Woodrow主席（对J.P.N.）的资助。

1. 样品制备
<ol><li>使用包含金箔和金网格支撑的网格16 （见 材料表），因为倾斜的数据收集会突出光束引起的运动17。 注意：对于本研究，在加湿（大于80%）的冷藏室（~4°C）中使用手动浸入和印迹技术18 对网格上的样品进行玻璃化。</li>
	<li>除非绝对必要，否则避免使用包含铜支架和碳箔的网格或连续的非晶碳?...

<ol>
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(180), e62765 (2022).</li><li>Patel, A., Toso, D., Litvak, A., Nogales, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+graphene+oxide+coating+improves+cryo-EM+sample+preparation+and+data+collection+from+tilted+grids.">Efficient graphene oxide coating improves cryo-EM sample preparation and data collection from tilted grids.</a> bioRxiv. , (2021).</li><li>Naydenova, K., Peet Mathew, J., Russo Christopher J, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifunctional+graphene+supports+for+electron+cryomicroscopy.">Multifunctional graphene supports for electron cryomicroscopy.</a> Proceedings of the National Academy of Sciences. 116 (24), 11718-11724 (2019).</li><li>Naydenova, 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=CryoEM+at+100+keV:+a+demonstration+and+prospects.">CryoEM at 100 keV: a demonstration and prospects.</a> IUCrJ. 6 (6), 1086-1098 (2019).</li><li>Herzik, M. A., Gonen, T., Nannenga, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> cryoEM: Methods and Protocols. , 125-144 (2021).</li><li>Cash, J. N., Kearns, S., Li, Y., Cianfrocco, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+cryo-EM+using+beam-image+shift+at+200+keV.">High-resolution cryo-EM using beam-image shift at 200 keV.</a> IUCrJ. 7 (6), 1179-1187 (2020).</li><li>Peck, J. V., Fay, J. F., Strauss, J. 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由样品粘附在空气-水界面上引起的首选颗粒取向是使用冷冻电镜SPA4，5，6进行常规高分辨率结构测定的最后一个主要瓶颈之一。这里介绍的数据收集方案提供了一种易于实现的策略，用于改善数据集中粒子的方向分布。我们注意到，该协议不需要额外的设备或软件，也不会影响数据收集速度。在倾斜样品的数据采集过程中，?...

在过去十年中，直接电子探测器1，2，3的出现刺激了使用单粒子冷冻电镜4，5，6解决的大分子和大分子组件的高分辨率结构的数量呈指数级增长。几乎所有纯化的大分子物质都有望使用冷冻电镜进行结构测定，除了最小的蛋白质~10 kDa大小或小于7。网格制备和结构测定所需的起始材料量至少比其他结构测定技术（如核磁共振波谱和X射线晶体学4，5，6）少一个数量级。
然而，通过冷冻电镜测定结构的主要挑战涉及合适的成像网格准备。一项使用不同玻璃化策略和网格评估不同样品的广泛研究表明，在冷冻电镜网格上玻璃化样品的大多数方法都会导致大分子优先粘附在空气-水界面上8。这种依从性可能导致四个次优结果：（1）大分子样品完全变性，在这种情况下，不可能成功的数据收集和处理;（2）样品部分变性，在这种情况下，有可能从未受损的大分子区域获得结构见解;（3）样品保留了天然结构，但图像中仅表示一组相对于电子束方向的粒子取向;（4）样品保留了天然结构，并且图像中表示了相对于电子束方向的一些但不是全部可能的粒子取向。对于情况（3）和（4），倾斜数据收集将有助于最大限度地减少影响重建冷冻电镜图的方向分辨率各向异性，并为各种样品提供可推广的解决方案9。从技术上讲，倾斜也可以使情况（2）受益，因为变性可能发生在空气 - 水界面处，并且同样限制了数据中表示的不同方向的数量。数据集中方向偏差的程度可以通过试验溶液添加剂来改变，但缺乏广泛的适用性阻碍了这些试错法。通过改变成像实验9 的几何形状，以单个优化的倾斜角度倾斜样品台足以改善方向分布（图1）。由于优先取向样品相对于电子束的几何配置，对于每个优先取向的簇，倾斜网格会生成相对于团簇质心的照明角度锥。因此，这分散了视图，从而改善了傅里叶空间采样和方向分辨率的各向同性。
在实践中，倾斜舞台有一些危害。倾斜标本载物台会在视场上引入焦点梯度，这可能会影响对比度传递函数 （CTF） 估计的准确性。倾斜的数据收集还可能导致光束诱导的粒子运动增加，这是由于对倾斜标本进行成像时电荷效应增加引起的。网格倾斜也会导致表观冰厚度增加，这反过来又导致显微照片嘈杂，并可能最终影响重建的分辨率5，9，10。通过应用协议和讨论部分中简要描述的高级计算数据处理方案，可以克服这些问题。最后，倾斜会导致粒子重叠增加，从而阻碍后续的图像处理管道。虽然这可以通过优化网格颗粒浓度在一定程度上缓解，但它仍然是一个重要的考虑因素。这里描述了一种易于实现的协议，用于使用Leginon软件套件（一种自动图像采集软件）进行倾斜数据收集，该套件可开放访问并与各种显微镜11，12，13，14兼容。该方法至少需要版本 3.0 或更高版本，版本 3.3 及更高版本包含专门的改进，以实现倾斜数据收集。此协议不需要额外的软件或设备。有关计算基础结构和安装指南的大量说明在别处提供15.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cryosparc Live v3.1.0+210216</td>
			<td>Structura Biotechnology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DPS protein</td>
			<td></td>
			<td></td>
			<td>Purification adapted from protocol described in K.Naydenova et al IUCrJ. 2019 Nov 1; 6(Pt 6): 1086&#8211;1098.</td>
		</tr>
		<tr>
			<td>K2 Summit Direct Electron Detector</td>
			<td>Gatan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leginon software suite</td>
			<td></td>
			<td></td>
			<td>C Suloway et al Journal of Structural Biology 151 (1): pp. 41-60.</td>
		</tr>
		<tr>
			<td>Manual plunging device</td>
			<td></td>
			<td></td>
			<td>Homemade guillotine-like device for vitrification of EM grids</td>
		</tr>
		<tr>
			<td>Talos Arctica</td>
			<td>FEI/Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UltrAufoil R1.2/1.3 300 mesh grids</td>
			<td>Quantifoil</td>
			<td>N1-A14nAu30-01</td>
			<td></td>
		</tr>
	</tbody>
</table>

single-particle cryo-em data collection with stage tilt using leginon

通过冷冻电子显微镜（cryo-EM）进行的单颗粒分析（SPA）现在是高分辨率结构生物学的主流技术。SPA的结构测定依赖于获得在薄冰层内玻璃化的大分子物体的多个不同视图。理想情况下，均匀分布的随机投影方向的集合将相当于物体的所有可能视图，从而产生以各向同性方向分辨率为特征的重建。然而，在现实中，许多样品都受到粘附在空气-水界面上的优先取向颗粒的影响。这导致数据集中的角度取向分布不均匀，重建中出现不均匀的傅里叶空间采样，从而转化为以各向异性分辨率为特征的地图。倾斜试样载物台提供了一种可推广的解决方案，通过提高取向分布的均匀性，从而改善傅里叶空间采样的各向同性，克服分辨率各向异性。该协议描述了使用Leginon（一种用于自动图像采集的软件）的倾斜阶段自动数据收集策略。该程序易于实施，不需要任何额外的设备或软件，并且与用于生物大分子成像的大多数标准透射电子显微镜（TEM）兼容。

使用0.3 mg/mL的DPS来演示0°、30°和60°倾斜时的成像。来自不同倾斜角度的数据收集在同一网格上不同网格区域。适合较高角度倾斜的CTF分辨率往往较差，就像在本研究中比较三个数据集时一样。 图4 显示了比较代表性图像和2D分类平均值。尽管在不同的倾斜角度下蛋白质浓度保持不变，但较高的倾斜角度使成像区域在颗粒浓度方面显得更加拥挤。这对于数据处理来说可能是个...

Watch this Scientific Journal Video about Single-Particle Cryo-EM Data Collection with Stage Tilt using Leginon at JoVE.com

Single-Particle Cryo-EM Data Collection with Stage Tilt using Leginon

本协议描述了冷冻电镜实验中倾斜单颗粒数据收集的通用且易于实施的方案。对于由于粘附空气-水界面而遭受优先方向偏差的样品，该程序对于获得高质量的EM图特别有用。

使用Leginon进行载物台倾斜的单颗粒冷冻电镜数据收集

Single-particle analysis (SPA) by cryo-electron microscopy (cryo-EM) is now a mainstream technique for high-resolution structural biology. Structure ...

倾斜样品台提供了一种简单且可推广的方法，可以在单颗粒冷冻电镜数据收集期间增加颗粒的方向分布。由于样品台倾斜而获得的颗粒的均匀分布可以提高整体地图质量和可解释性。首先对准显微镜，以确保标本的平行照明并最大限度地减少彗差。要确定适合数据收集的方块，请记录一个没有载物台倾斜或带载物台倾斜的网格图集，该图集提供了整体网格质量的概述，并初步指示了适合数据收集的区域。在正方形采集节点中使用的放大倍率下手动检查正方形。寻找箔片完好无损、看起来不脱水且具有理想冰厚度的正方形，并将标本载物台移动到感兴趣的正方形。使用载物台倾斜约15度的α摆动器确定载物台位置的共心高度，并使用显微镜的键盘面板调整Z高度以使载物台达到共心高度。确保在 Alpha 摆动例程期间图像偏移最小。要找到更准确的 Z 高度，请在聚焦器节点中以方形采集节点中使用的放大倍率估计它。调整聚焦器节点的设置，并在正方形初始排队期间启用或禁用精细 Z 焦点选项。然后按模拟。将样品载物台倾斜到所需的倾斜角度，以便在真正的真心高度收集数据，并在必要时将载物台重新居中。在方形采集节点中按模拟，开始排队整个采集节点曝光的目标。接下来，选择一个Z焦点目标和具有适合高倍率曝光的孔的区域，并将目标提交到队列进行成像。将标本载物台恢复到未倾斜的状态，然后移动到下一个正方形。重复这些步骤，直到排队的整个曝光次数足够多。所有方块排队后，转到整个目标节点，然后按提交排队目标。在节点设置中选择允许用户验证所选目标，然后手动检查高倍率曝光采集节点选择的目标，以测试当标本载物台倾斜时，自动EM孔查找器是否可以准确识别适合图像采集的区域。用户对定位准确性感到满意后，取消选择允许用户验证所选目标选项以自动收集数据。此处显示了在方形放大倍率下收集的不同倾斜角度的网格的代表性图像，这些图像收集的齐心Z高度附近和远处。光束的中心由红色同心环的中心表示，绿色箭头表示感兴趣的正方形。在感兴趣的正方形旁边有一个断开的格网要素以供参考。此处显示的是 0 度、30 度和 60 度倾斜角度下收集的代表性孔暴露和 2D 类平均值。尽管在不同的倾斜角度下蛋白质浓度保持不变，但较高的倾斜角度使成像区域在颗粒浓度方面显得更加拥挤。受过度拥挤影响的 2D 类平均值显示在此红色框中。该方案的简单性使显微镜的任何标准用户都可以轻松调整倾斜的数据收集策略。

Research

JoVE Journal

Biochemistry

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

使用SPHIRE的电子冷冻显微镜图像的高分辨率单粒子分析

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

科研

生物化学

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

生物大分子晶体的完全自主表征及数据采集

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

Fixed Target Serial Data Collection at Diamond Light Source

金刚石光源的固定目标串行数据收集

Serial data collection is a relatively new technique for synchrotron users. A user manual for fixed target data collection at I24, Diamond Light Source is ...

Cryo-EM and Single-Particle Analysis with Scipion

使用沉降机进行冷冻电镜和单颗粒分析

Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at ...

Single Particle Cryo-Electron Microscopy: From Sample to Structure

单颗粒冷冻电子显微镜：从样品到结构

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The ...

Preparation of High-Temperature Sample Grids for Cryo-EM

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, ...

A Robust Single-Particle Cryo-Electron Microscopy (cryo-EM) Processing Workflow with cryoSPARC, RELION, and Scipion

A Robust Single-Particle Cryo-Electron Microscopy cryo-EM Processing Workflow with cryoSPARC, RELION, and Scipion

强大的单颗粒冷冻电子显微镜 （cryo-EM） 处理工作流程，具有冷冻 SPARC、RELION 和 Scipion

Recent advances in both instrumentation and image processing software have made single-particle cryo-electron microscopy (cryo-EM) the preferred method ...

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope

使用200 KV透射电子显微镜常规收集高分辨率冷冻电镜数据集

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ...

The Peel-Blot Technique: A Cryo-EM Sample Preparation Method to Separate Single Layers From Multi-Layered or Concentrated Biological Samples

剥离印迹技术：一种冷冻电镜样品制备方法，用于从多层或浓缩生物样品中分离单层

The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers ...

Accelerating Macromolecular Structural Analysis Using Smart Leginon Autoscreen

Cryo-Electron Microscopy Screening Automation Across Multiple Grids Using Smart Leginon

Author Spotlight: Enhancing Cryo-Electron Microscopy by Automated Data Collection and Analysis Techniques

Advancements in cryo-electron microscopy (cryoEM) techniques over the past decade have allowed structural biologists to routinely resolve macromolecular ...