Name: user-friendly, high-throughput, and fully automated data acquisition software for single-particle cryo-electron microscopy
Uploaded: 2021-07-29T14:45:00
Description: Watch this Scientific Journal Video about User-friendly, High-throughput, and Fully Automated Data Acquisition Software for Single-particle Cryo-electron Microscopy at JoVE.com

我们感谢印度生物技术部，科学技术部（DST）和科学部以及人力资源开发部（MHRD）提供资金和IISc-Bangalore的冷冻电镜设施。我们感谢位于班加罗尔IISc的国家冷冻电镜设施的DBT-BUILDER计划（BT / INF / 22 / SP22844 / 2017）和DST-FIST（SR / FST / LSII-039 / 2015）。我们感谢科学与工程研究委员会（SERB）（拨款编号-SB/S2/RJN-145/2015、SERB-EMR/2016/000608和SERB-IPA/2020/000094）、DBT（拨款编号：SERB）的财政支持。BT/PR25580/BRB/10/1619/2017）。我们感谢Ishika Pramanick女士准备冷冻电镜网格，冷冻电镜数据收集和准备 材料表。我们还感谢Suman Mishra先生的冷冻电镜图像处理，并帮助我们准备数字。我们感谢Raghavan Varadarajan教授帮助我们为这项研究获得纯化的刺突蛋白样品。

注：冷冻电镜数据收集需要三个重要步骤：1.冷冻电镜网格制备，2.显微镜的校准和对准，3.自动数据收集（图1）。此外，自动数据收集细分为a.合适区域选择，b.Latitude-S优化，c.启动自动孔选择和d.启动自动数据采集（图1）。
1. 用于自动数据采集的冷冻电镜网格制备和样品加载
<ol><li>使用辉光放电器清洁网格，并根据实验要求改?...

<ol>
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Latitude-S 是一个直观的用户界面，它提供了一个环境，可以在两天内自动设置和收集数千张高分辨率显微照片或电影文件。它提供了跨网格的轻松导航，并在显微镜载物台从低放大倍率移动到高放大倍率时保持显微镜载物台的位置。使用Latitude-S进行数据采集的每个步骤都非常省时，具有简单的用户界面，以高达4.5 GB / s的速度快速流式传输数据以及在采集过程中同时显示数据等功能。此外，预校准...

单颗粒冷冻电镜已成为生物大分子高分辨率结构测定的主流结构生物学技术1。单颗粒重建依赖于获取大量玻化样品的显微照片来提取二维（2D）颗粒图像，然后用于重建生物大分子的三维（3D）结构2，3。在DED开发之前，单粒子重建获得的分辨率在4到30 Å4，5之间。最近，单颗粒冷冻电镜可实现的分辨率已超过1.8 Å6。DED和自动数据采集软件是这一分辨率革命7的主要贡献者，其中人为数据收集干预最少。通常，冷冻电镜成像以低电子剂量率（20-100 e / Å2）进行，以最大限度地减少电子束引起的生物样品辐射损伤，这有助于图像中的低信噪比（SNR）。这种低SNR阻碍了使用单颗粒分析表征生物大分子的高分辨率结构。
新一代电子探测器是基于CMOS（互补金属氧化物半导体）的探测器，可以克服这些与SNR相关的低障碍。这些直接检测CMOS相机可以快速读出信号，因此相机为生物大分子提供了更好的点扩散功能，合适的SNR和出色的检测量子效率（DQE）。直接检测相机在记录的图像中提供高SNR8 和低噪声，从而定量提高检测量子效率（DQE） - 测量探测器向图像增加的噪声量。这些相机还以每秒数百帧的速度录制短片，从而实现快速数据采集9，10。所有这些特性使得快速直接检测相机适用于低剂量应用。
运动校正堆栈图像用于数据处理，以计算2D分类，并使用各种软件包（如RELINE11，FREALIGN12，cryoSPARC13，cisTEM14和EMAN215）重建大分子的3D密度图。然而，对于单粒子分析，需要一个巨大的数据集来实现高分辨率结构。因此，自动数据采集收费对于数据收集至关重要。为了记录大型冷冻电镜数据集，在过去十年中已经使用了几个软件包。专用软件包，如AutoEM16，AutoEMation17，Leginon18，SerialEM19，UCSF-Image420，TOM221，SAM22，JAMES23，JADAS24，EM-TOOLS和EPU，已经开发用于自动数据采集。
这些软件包使用常规任务，通过将低倍率图像与高倍率图像相关联来自动查找孔位置，这有助于识别具有专有冰厚的玻璃体冰的孔，以便在低剂量条件下进行图像采集。这些软件包通过连续几天获取大量高质量数据，减少了重复性任务的数量，并提高了冷冻电镜数据收集的吞吐量，没有任何中断和操作员的物理存在。Latitude-S是一个类似的软件包，用于单颗粒分析的自动数据采集。但是，该软件包仅适用于K2 / K3 DED，并随这些探测器一起提供。
该协议证明了Latitude-S在使用配备200 keV冷冻电镜的直接电子探测器自动图像采集SARS-CoV-2刺突蛋白方面的潜力（见 材料表）。利用该数据采集工具，自动采集3000个SARS-CoV-2刺突蛋白电影文件，并进行进一步的数据处理，得到分辨率为3.9-4.4Å的刺突蛋白结构。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Blotting paper</td>
			<td>Ted Pella, INC.</td>
			<td>47000-100</td>
			<td>EM specimen preparation item</td>
		</tr>
		<tr>
			<td>Capsule</td>
			<td>Thermo Fisher Scientific</td>
			<td>9432 909 97591</td>
			<td>EM specimen preparation unit</td>
		</tr>
		<tr>
			<td>Cassette</td>
			<td>Thermo Fisher Scientific</td>
			<td>1020863</td>
			<td>EM specimen preparation unit</td>
		</tr>
		<tr>
			<td>C-Clip</td>
			<td>Thermo Fisher Scientific</td>
			<td>1036171</td>
			<td>EM specimen preparation item</td>
		</tr>
		<tr>
			<td>C-Clip Insertion Tool</td>
			<td>Thermo Fisher Scientific</td>
			<td>9432 909 97571</td>
			<td>EM specimen preparation tool</td>
		</tr>
		<tr>
			<td>C-Clip Ring</td>
			<td>Thermo Fisher Scientific</td>
			<td>1036173</td>
			<td>EM specimen preparation item</td>
		</tr>
		<tr>
			<td>EM grid (Quantifoil)</td>
			<td>Electron Microscopy Sciences</td>
			<td>Q3100AR1.3</td>
			<td>R 1.2/1.3 300 Mesh, Gold</td>
		</tr>
		<tr>
			<td>Glow discharge Machine</td>
			<td>Quorum</td>
			<td>N/A</td>
			<td>Quorum GlowQube glow discharge machine</td>
		</tr>
		<tr>
			<td>K2 DED</td>
			<td>Gatan Inc.</td>
			<td>N/A</td>
			<td>Cryo-EM data collection device (Camera)</td>
		</tr>
		<tr>
			<td>Latitude S Software</td>
			<td>Gatan Inc.</td>
			<td></td>
			<td>Imaging software</td>
		</tr>
		<tr>
			<td>Loading station</td>
			<td>Thermo Fisher Scientific</td>
			<td>1130698</td>
			<td>EM specimen preparation unit</td>
		</tr>
		<tr>
			<td>Talos 200 kV Arctica</td>
			<td>Thermo Scientific&#8482;</td>
			<td>N/A</td>
			<td>Cryo-Electron Microscope</td>
		</tr>
		<tr>
			<td>Vitrobot Mark IV</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td>EM specimen preparation unit</td>
		</tr>
	</tbody>
</table>

user-friendly, high-throughput, and fully automated data acquisition software for single-particle cryo-electron microscopy

近年来，单颗粒冷冻电镜（cryo-EM）的技术和方法学进步为生物大分子的高分辨率结构测定铺平了新的途径。尽管冷冻电镜取得了显著进展，但在单颗粒分析工作流程的各个方面仍有改进的余地。单颗粒分析需要合适的软件包来实现高通量自动数据采集。在过去八年中，开发了几个自动数据采集软件包，用于单颗粒冷冻电镜的自动成像。本文提出了一种全自动图像采集管线在低剂量条件下用于玻化生物分子的应用。 
它演示了一个软件包，可以完全、自动、精确地收集冷冻电镜数据。此外，该软件包可以轻松控制各种微观参数。该协议证明了该软件包在配备直接电子探测器（DED）的200 keV冷冻电子显微镜对严重急性呼吸系统综合征冠状病毒2（SARS-CoV-2）刺突蛋白进行自动成像方面的潜力。在单次（48小时）的数据收集中采集了大约3，000张冷冻电镜电影图像，产生了SARS-CoV-2刺突蛋白的原子分辨率结构。此外，该结构研究表明，刺突蛋白采用两种主要构象，1-RBD（受体结合结构域）向上开放，所有RBD向下闭合构象。

在当前的大流行形势下，冷冻电镜在表征SARS-CoV-226，27，28，29的各种蛋白质的结构方面起着关键作用，这可能有助于开发针对该病毒的疫苗和药物。迫切需要以有限的人力资源进行快节奏的研究工作，以抗击2019年的冠状病毒病。单颗粒冷冻电镜中的数据采集是大分子结构测定中耗时但至关重要的步骤...

Watch this Scientific Journal Video about User-friendly, High-throughput, and Fully Automated Data Acquisition Software for Single-particle Cryo-electron Microscopy at JoVE.com

User-friendly, High-throughput, and Fully Automated Data Acquisition Software for Single-particle Cryo-electron Microscopy

单颗粒冷冻电子显微镜需要合适的软件包和用户友好的管道来实现高通量自动数据采集。在这里，我们介绍了全自动图像采集软件包Latitude-S的应用，以及用于在低剂量条件下收集玻化生物分子数据的实用管道。

用户友好型、高通量和全自动数据采集软件，用于单颗粒冷冻电子显微镜

In the past several years, technological and methodological advancements in single-particle cryo-electron microscopy (cryo-EM) have paved a new avenue for ...

自动数据采集软件对于收集大量冷冻数据以进行生物无大分子的结构表征是非常必要的。这里使用自动数据采集软件包来表征与病原体生物学直接相关的各种生物大无分子的结构。例如，分枝杆菌，HIV和SASCO 2。首先，通过单击"开始"菜单中的数字显微图来启动Latitude-S自动数据采集软件。接下来，选择"技术管理器"，然后选择"纬度-S"图标，用于"单粒子自动数据收集"。要基于以前的会话设置创建会话，请选中调色板中的"基于以前的会话"复选框，然后选择新按钮或继续现有会话，请按"继续"按钮。要启动全新的会话，请单击选项板中的新选项卡。选择要继续的会话的文件夹，然后选择要保存数据的文件夹。接下来，单击设置图标，然后在出现的"托管状态浏览"框中，添加状态，设置TEM条件，相机条件以及图像或库存选项。然后，命名状态或打开状态设置摘要以打开保存设置。接下来，通过单击 Atlas 状态调色板配置 Atlas 状态，并设置放大倍率、照明条件和相机曝光时间的参数，然后单击下一步移动到下一个状态。然后通过单击网格状态调色板来配置网格状态，设置显微镜成像光学元件，照明条件和相机曝光时间的参数，然后单击下一步。通过单击孔调色板配置孔状态，并设置成像光学元件、照明条件、分档和相机曝光时间参数。单击"下一步"后，通过单击对焦调色板配置下一个对焦状态，并设置成像光学元件、照明条件、合并和相机曝光时间的显微镜设置。然后聚焦在孔附近的无定形碳区域，然后单击"下一步"移动到下一个状态。通过单击数据调色板配置数据状态并设置显微镜设置的参数，然后单击下一步。单击焦点配置调色板，在给定选项卡中指定散焦值的范围和步长，然后按下一步按钮进入下一步。将焦点放在网格上所需的功能上，然后单击捕获按钮。然后将红叉标记定位在不同状态的每个图像上的同一特征上。从"焦点"、"数据"和"洞"状态开始，因为视野大于"图集"和"网格"状态。接下来，缩放地图集和格网状态以将红叉标记定位在同一要素上。要计算五种不同状态之间的位置和偏移，并将位置和偏移量反映到输出窗口中，请单击计算按钮。单击捕获调色板，然后根据要求选择 Atlas 的大小以覆盖整个网格或部分网格。要捕获地图集，请在地图集上进行导航，然后根据冰的厚度选择网格正方形。选择所需的网格方块后，单击计划按钮，并在捕获每个网格方块时观察网格方块中的磁贴填满。单击计划按钮后，通过添加孔的位置在网格正方形中选择一个具有代表性的孔。获取孔图像后，定义数据和焦点位置，并将布局另存为模板。单击"自动查找"，输入孔大小，然后单击程序中的"查找"按钮以根据直径自动查找孔。设置强度以从网格方块和冰污染中移除孔。通过自动查找添加所选和黄色标记工具后，单击纬度任务中的计划按钮。根据使用cisTEM软件对运动校正显微照片的初始手动筛选，发现大多数数据都在所需的信号范围内。此外，在散焦范围内收集的图像也通过cisTEM手动检查。手动选择尖峰颗粒以计算结构可视化的2D类平均值，这强烈建议使用各自的数据集进行高分辨率结构表征。3D分类表明，Spike蛋白在向上开盘确认中具有1RBD，在向下收盘确认中具有所有其他RBD。使用C1对称性重建了Spike蛋白的1RBD向上开放确认。尖峰蛋白图在侧面顶部和底部视图中表示。EM图具有原子结构，可以更好地可视化侧链。RBD下行收盘确认通过C3对称性进行了细化，尖峰3D细化模型和EM图，并拟合了如图所示的原子结构。被鉴定为S2子结构域的Spike蛋白的中间确认表明单个氨基酸残基的侧链。结果表明，Latitude-S可以收集生物大分子的高分辨率冷冻电镜数据。该自动数据收集软件可用于任何其他电子显微镜系统以自动收集数据。

Research

JoVE Journal

Immunology and Infection

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

使用低温电子断层扫描和自动子断层扫描平均HIV包膜糖蛋白分子结构的测定

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

科研

免疫与感染

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

HIV - 1衣壳大会通过低温电子显微镜的结构和迭代螺旋实时空间重构

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

High-throughput Detection Method for Influenza Virus

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

定量高通量单细胞的细胞毒性T细胞的测定

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

高通量筛选RNA病毒的广谱化学抑制剂

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

在参与宿主因素的高通量筛选基于显微镜的检测方法<em&gt;布鲁氏菌</em&gt; Hela细胞感染

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

A High-throughput Shigella-specific Bactericidal Assay

A High-throughput Shigella-specific Bactericidal Assay

一种高通量志贺氏菌特异性杀菌试验

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...