我们感谢D. Roderer为我们提供了TcdA1显微照片。我们感谢Steve Ludtke持续支持EMAN2基础设施。这项工作得到了马克斯·普朗克学会（SR）和欧洲理事会根据欧盟第七框架计划（FP7 / 2007-2013）（授权号615984）（授予SR）的资助，并获得了美国国家研究机构健康R01 GM60635至PAP）。

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作者宣称他们没有竞争的经济利益。

单粒子低温电动机近年来呈现出快速发展的趋势，并提供了许多具有重要生物学意义的大分子复合物的原子分辨率结构25 。为了支持当前正在进入该领域的大量新手用户，我们开发了单粒子图像分析平台SPHIRE ，并为此展示了包括电影对齐，粒子采集，CTF估计，初始模型在内的整个工作流程的漫游协议计算，2D和3D异质性分析，高分辨率3D细化和局部分辨率估计和滤波。 这里描述的协议旨在作为使用感兴趣的蛋白质的cryo-EM显微照片以及由SPHIRE的独立GUI提供的计算工具的帮助的3D结构确定的简短指南。 工作流的主要特点是大部分的程序只需要运行一次，因为它们依赖于通过重复性19进行验证的概念，并且不需要参数调整。这种自动验证机制是SPHIRE与其他软件包的主要优点，因为结果往往是客观的和可重复的，最重要的是可以以可接受的计算成本获得。该管道还为经验丰富的用户提供了丰富的诊断信息，以自己的方法进行进一步的独立验证和评估。然而，在结构生物学和电子显微镜中至少具有元素理论背景的新手用户应该能够使用自己的数据和自动验证程序获得近原子分辨率结构。 然而，获得近原子分辨率结构并不总是直接的，结果将高度取决于样本的质量和输入数据一个。对于这里给出的程序，假设有足够数量的高质量未对齐的原始EM电影可用，其平均值显示清晰可辨的均匀和随机取向的单个颗粒。通常，对于分子的对称性，大小或整体形状没有限制，但是低分子量可能是限制因素，特别是当蛋白质具有无特征的球形形状时。通常，具有高点组对称性的较大，有序粒子的分析要求较低。因此，强烈建议新手用户首先使用精确表征的cryo-EM数据集来运行本协议。 SPHIRE教程数据（http:/sphire.mpg.de）或EMPIAR提交的数据集之一（https://www.ebi.ac.uk/pdbe/emdb/empiar/）与原始电影是一个很好的起点。 在处理自己的数据时，很可能某些数据集或某些图像不能满足某些要求标准。在这方面，除了由程序对工作流程的主要步骤执行的自动化稳定性和再现性检查之外，仍然建议用户在协议的某些"检查点"处目视检查结果，特别是如果最终重建不满意在电影对准（ 协议步骤2 ）和CTF估计（ 协议步骤3 ）之后，可以在显微照片级别进行第一次视觉检查。所得到的运动校正平均值应显示清晰可辨和分离好的单个颗粒，其功率谱应显示清晰可辨的各向同性Thon环。它们可见的空间频率在大多数情况下定义了最终决定结构原则上最高的分辨率。具有足够质量的运动校正平均值及其功率谱的示例显示在＆＃34;代表性的结果"。可以借助SPHIRE的Drift和CTF评估GUI工具（http://sphire.mpg.de/wiki/doku.php），去除可能对最终结果产生负面影响的异常图像。 关于粒子筛选，SPHIRE流程中的关键步骤是使用ISAC的2D分类（ 协议步骤5.2） 。这里，用户应该控制由程序自动识别的可再现的2D类平均采用足够准确地均匀地覆盖角度空间的取向范围。如果课程平均质量不令人满意（嘈杂和/或模糊的图像）和/或可再现的班级数量非常低，考虑提高自动采摘质量，优化数据集成像或样品制备。在大多数情况下，不可能从不产生良好2D类别平均值的数据集中计算可靠的重构。高品质2D课程的例子"代表性结果"一节中显示了愤怒。 需要至少100级平均值以自动方式获得可靠的初始3D模型（ Protocol步骤6.1 ）。对于此步骤，用户应选择具有最高质量的平均值，并包括尽可能多的不同粒子取向。初始模型的质量对于随后的高分辨率3D细化的成功至关重要。 在其他软件包中，有时执行3D分类以去除"坏"粒子8,9 。然而，在SPHIRE中，大多数这些粒子已经在使用ISAC的2D分类期间被自动消除。因此，如果重建和3D变异性分析指示数据集的异质性，则建议仅执行计算密集的3D排序步骤。 最重要的是，用户应该仔细仔细检查所得到的3D体积（ 协议步骤9.3 ），并确认相应密度的特征与标称分辨率一致。分辨率&lt;9Å，对应于α-螺旋的棒状密度变得可见。在分辨率&lt;4.5Å时，对应于β-折叠中的链的密度通常很好地分离并且体积大的氨基酸变得可见。高分辨率图（&lt;3Å）应显示清晰可辨的侧链，从而允许构建准确的原子模型。 迄今获得的结果表明，在SPHIRE的自动重现性测试和最小目视检查的帮助下，本协议通常适用于任何类型的单粒子低温 - EM项目。显示每个处理步骤的代表性结果，用于重建TcdA1毒素Photorhabdus luminescens 21 ，已被解决为近原子分辨率。类似质量的密度图可用于通过从头主干跟踪以及相互或实际空间细化构建可靠的原子模型，从而为理解复杂的分子机制提供坚实的结构框架。 访问代码: EM结构和未处理电影的坐标已分别存放在电子显微镜数据库和电子显微镜导频图像存档中，登录号为EMD-3645和EMPIAR-10089。

直接电子探测器技术的发展，单粒子低温电动机的显着进步正在重塑结构生物学1 。与X射线晶体学相比，该技术只需要少量的蛋白质材料，而不需要结晶，同时对样品的纯度提出更少的限制，并且仍允许测定近原子分辨率下的结构。重要的是，不同的组成或状态现在可以在计算上分离，不同构象的结构确定可以以前所未有的细节进行。最近，挑战性分子的密度图可以通过允许从头模型建立的决议产生，从而深入了解其作用模式2,3,4,5。 3DEM（3D电子显微镜）社区（https://en.wikibooks.org/wiki/Software_Tools_For_Molecular_Microscopy）中提供了各种各样的图像处理软件包，大多数都在不断的开发中。已经通过几种不同的软件包，包括EMAN2 6 ，IMAGIC 7 ，FREALIGN 8 ，RELION 9 ，SPIDER 10和SPARX 11 ，显示各种分子量和对称性的蛋白质已经实现了近原子分辨率。每个包需要不同级别的用户专业知识，并提供不同级别的用户指导，自动化和可扩展性。此外，虽然一些程序提供了完整的环境来促进图像分析的所有步骤，但是其他程序被设计为优化特定任务，例如从已知的r开始的对准参数的改进参考结构。最近，已经开发了几个平台，包括APPION 12和SCIPION 13 ，它提供了一个整合来自上面列出的不同软件包的方法和协议的单一处理流程。 为了有助于目前低温EM的发展，SPARX被重新开发成为一个新的独立和完整的单粒子分析平台，称为SPHIRE（用于高分辨率电子显微镜的SPARX）。为了增加该领域新研究者的技术的可及性，并且为了应对现代全自动高端电子显微镜产生的大量数据，通过引入易于使用的方法重新设计和简化了处理管道图形用户界面（GUI），并自动执行工作流程的主要步骤。此外，添加了新的算法以允许从cr获得快速，可重现和自动化的结构确定yo-EM图像。此外，引入了通过重现性的验证，以避免在细化和异质性分析期间产生的常见伪像。 虽然该程序被广泛修改，但它的核心功能得到了维护:简单的开源代码，现代面向对象的设计和所有基本功能的Python接口。因此，它没有改变为黑盒子程序，使用户能够学习和轻松地修改Python代码，创建其他应用程序或修改整个工作流程。这对于非标准的低温电力项目尤其有用。 这里我们提出一种使用SPHIRE的GUI从cryo-EM图像获得近原子分辨率密度图的协议。它详细描述了从原始的cryo-EM直接检测器电影生成密度图所需的所有步骤，并不限于任何特定的大分子类型。这个协议主要是指引newc通过工作流程在实地工作，并提供关于处理的关键步骤以及一些可能的陷阱和障碍的重要信息。更高级的功能和SPHIRE背后的理论背景将在其他地方描述。

<table border="0" cellpadding="0" cellspacing="0" width="790">
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	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">SPHIRE</td>
			<td style="width:288px;">Max Planck Institute of Molecular Physiology- Dortmund&#160; and Houston Medical School, Houston, Texas&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">http://sphire.mpg.de</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UCSF Chimera</td>
			<td style="width:288px;">University of California, San Francisco</td>
			<td style="width:119px;"></td>
			<td>http://www.cgl.ucsf.edu/chimera/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Unblur</td>
			<td style="width:288px;">Janelia Farm Research Campus, Ashburn</td>
			<td style="width:119px;"></td>
			<td>http://grigoriefflab.janelia.org/unblur</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Coot</td>
			<td style="width:288px;">MRC Laboratory of Molecular Biology,&#160; Cambridge</td>
			<td style="width:119px;"></td>
			<td>http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EMAN2</td>
			<td style="width:288px;">Baylor College of Medicine, Houston</td>
			<td style="width:119px;"></td>
			<td>http://blake.bcm.edu/emanwiki/EMAN2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Computing Cluster with 1824 cores</td>
			<td style="width:288px;">Max Planck Institute of Molecular Physiology</td>
			<td style="width:119px;"></td>
			<td>Linux Cluster with 76&#160; nodes, each with 2 Processors Xeon E5-2670v3 12C 2.30 GHz and 128 Gb RAM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TITAN KRIOS electron microscope&#160;</td>
			<td style="width:288px;">FEI</td>
			<td style="width:119px;"></td>
			<td>300 kV, Cs correction, XFEG</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Falcon II direct electron detector</td>
			<td style="width:288px;">FEI</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EPU (automated data acquisition software)</td>
			<td style="width:288px;">FEI</td>
			<td style="width:119px;"></td>
			<td>https://www.fei.com/software/epu/</td>
		</tr>
	</tbody>
</table>

high-resolution single particle analysis from electron cryo-microscopy images using sphire

SPHIRE（用于高分辨率电子显微镜的SPARX）是用于半自动处理单粒子电子冷冻显微镜（cryo-EM）数据的新型开源，用户友好的软件套件。这里介绍的协议详细介绍了如何通过引导用户通过单个粒子结构确定管道的所有步骤从cryo-EM显微照片电影开始获得近原子分辨率结构。这些步骤由新的SPHIRE图形用户界面进行控制，并需要最少的用户干预。使用该方案，来自Photorhabdus luminescens的TcdA1（ Tc毒素复合物）的结构来自仅9500个单一颗粒。这种简化的方法将帮助没有广泛的处理经验和先验结构信息的新手用户获得其纯天然状态的纯化大分子复合物的无噪声和无偏倚的原子模型。

<html>从Photorhabdus发光体 Tc复合体（TcdA1） 20,21,22的A分量的112个直接检测器电影开始执行上述协议。将该数据集记录在具有高亮度场致发射枪（XFEG）的Cs校正电子冷冻显微镜上，其在300kV的加速电压下工作。以样品量表上的像素尺寸为1.14Å的总剂量为60 e - /Å -2自动获取图像。在电影帧对准（ 协议步骤2 ）之后，所得到的运动校正平均值具有扩展到高分辨率的各向同性Thon环（ 图2a ）。单个颗粒容易可见并且分离良好（ 图2b ）。然后使用e2boxer的群组工具挑选粒子lass ="xref"&gt; 18（ 协议步骤4.1 ）。在这种情况下，使用更具选择性的选项设置适当的阈值（ 图2c ）。 112幅数字显微照片产生了9,652颗颗粒。大部分提取的图像（ 方案步骤4.2 ）包含明确定义的颗粒，并且它们的盒尺寸是粒径的约1.5倍，如推荐的（ 图2d ）。接下来，使用ISAC，进行2D异质性分析（ 方案步骤5 ）。它产生98个班级平均数（ 图3a ）。使用这些2D类平均值，使用VIPER（ 方案步骤6 ）以中间分辨率计算出一个从头模型（ 图3b ）。该模型与先前在3.9Å分辨率22 （ 图3c ）中解析的TcdA1的晶体结构显示出极好的一致性。这个从头模型被用作初始模式<html> （MERIDIEN），仅从〜40,000个不对称单位产生3.5（λ3.3标准）重建（ 方案步骤7 ）（ 图4 ）。这个近原子分辨率图是在24小时内获得的，使用多达96个CPU用于从多个内核中受益的工作流程。 对于3D变异性分析（协议步骤8），在步骤8.3.3中使用每组只有2,000个粒子图像（ 即 ，过程从5个初始3D组开始），并且在步骤8.3.4中由200个图像组成的最小组大小少量颗粒（〜10,000）。分析显示局部柔韧性主要在复合物的N末端区域，其含有用于纯化的His标签（ 图5a ）。事实上，在先前公布的TcdA1晶体结构中，12个N-末端残基和His标签未被解析"22，这个最可能无序的区域在目前的cryo-EM密度上仍未得到解决，这可能是由于其灵活性。在受体结合结构域和BC结合结构域处检测到另外的变异性（ 图5a ）结构的满意分辨率和数据集的相当小的尺寸，这种异质性被决定是可以容忍的，因此不执行聚焦的3D分类23。最后，计算最终密度图的局部分辨率（ 协议步骤9.1，图5b ），并且锐化的3D地图被本地过滤（ 协议步骤9.2） 。该质量的体积可以用于使用Coot 24或任何其他细化工具（ 图6 ）的从头模型构建。 <img alt="图1" src="/files/ftp_upload/55448/55448fig1.jpg" /> &lt;strong&gt;图1:使用SPHIRE的图像处理。 （ a ）SPHIRE软件包的GUI。可以通过选择GUI左侧的相应图标（"工作流步骤"）来激活工作流程的特定步骤。与该工作流程步骤相关联的命令和实用程序将显示在GUI的中央区域。选择其中一个命令后，相应的参数显示在GUI的右侧区域。高级参数通常不需要修改预设的默认值。 （ b ）使用SPHIRE GUI进行单粒子图像处理的工作流阶段。 <a href="http://ecsource.jove.com/files/ftp_upload/55448/55448fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" src="/files/ftp_upload/55448/55448fig2.jpg" /> 图2:运动校正和Partic提取。 （ a ， b ）在散焦为1.7μm时记录的典型高品质，低剂量，漂移校正数字显微镜。注意在功率谱（a）中分辨率为2.7埃的各向同性Thon环和2D图像（ b ）中的可辨别的颗粒。 （ c ）使用e2boxer进行粒子选择。绿色圆圈表示选定的粒子。 （ d ）从剂量加权显微照片提取的典型原始颗粒。比例尺= 20nm。 <a href="http://ecsource.jove.com/files/ftp_upload/55448/55448fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" src="/files/ftp_upload/55448/55448fig3.jpg" /> 图3:2D聚类和初始模型生成。 （ a ）2D类平均画廊，多数代表侧视图o<html> f粒子。比例尺= 20nm。 （ b ）使用RVIPER从无参考平均值中获取的TcdA1的从头开始的 3D地图。 （ c ）将TcdA1晶体结构（带）（pdb-id 1VW1）刚体拟合为初始低温 - EM密度（透明灰色）。 <a href="http://ecsource.jove.com/files/ftp_upload/55448/55448fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" src="/files/ftp_upload/55448/55448fig4.jpg" /> 图4:TcdA1的Cryo-EM三维结构。 （ a ， b ）使用〜9,500粒子图像计算的TcdA1的最终3.5Å密度图:（ a ）侧面和（ b ）顶视图。 （ c ）α螺旋和β片的低温 - EM密度的代表性领域。arge.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图5" src="/files/ftp_upload/55448/55448fig5.jpg" /> 图5:变异性分析和局部分辨率。 （ a ）锐化的TcdA1冷冻 - EM图（灰色）和变异性图（绿色）的表面。为了更好的清晰度，将可变性图低通滤波至30埃。 （ b ）根据局部分辨率（Å）对TcdA1进行锐化的低温电子地图的表面渲染。注意高变异性和低局部分辨率的区域之间的拓扑一致性。 <a href="http://ecsource.jove.com/files/ftp_upload/55448/55448fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图6" src="/files/ftp_upload/55448/55448fig6.jpg" /> 图6:3D Mod使用Coot构建TcdA1。低密度EM密度和原子模型的代表性区域显示为α-螺旋。原子模型是使用Coot 从头建造的。 <a href="http://ecsource.jove.com/files/ftp_upload/55448/55448fig6large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE at JoVE.com

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

本文介绍了使用软件套件SPHIRE处理低温EM图像的协议。目前的协议可以应用于几乎所有瞄准近原子分辨率的单粒子EM项目。

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 ...

high-resolution-single-particle-analysis-from-electron-cryo

Research

JoVE Journal

Biochemistry

50.2K 次观看.  Max Planck Institute of Molecular Physiology. 本文介绍了使用软件套件SPHIRE处理低温EM图像的协议。目前的协议可以应用于几乎所有瞄准近原子分辨率的单粒子EM项目。 

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

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

细菌显示文库的半自动化筛选肽亲和试剂的发现与分离结果分析

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

科研

生物化学

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

β-淀粉脂质诱导纤维蛋白斑点结构异常的光谱分析

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

单粒子冷冻电子显微镜在结构中测定脂质敏感分通道 trpc3 的表达与纯化

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.

A versatile cryoslicing BN-MS protocol using a microtome is presented for high-resolution complexome profiling.

通过低温BN-MS分析进行高分辨率复杂分析

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

Cryo-EM and Single-Particle Analysis with Scipion

Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at near-atomic resolution. In single-particle analysis, the vitrified sample is imaged by an electron beam and the detectors at the end of the microscope column produce movies of that sample. These movies contain thousands of images of identical particles in random orientations. The data need to go through an image processing workflow with multiple steps to obtain the final 3D reconstructed volume. The goal of the image processing workflow is to identify the acquisition parameters to be able to reconstruct the specimen under study. Scipion provides all the tools to create this workflow using several image processing packages in an integrative framework, also allowing the traceability of the results. In this article the whole image processing workflow in Scipion is presented and discussed with data coming from a real test case, giving all the details necessary to go from the movies obtained by the microscope to a high resolution final 3D reconstruction. Also, the power of using consensus tools that allow combining methods, and confirming results along every step of the workflow, improving the accuracy of the obtained results, is discussed.

Single-particle analysis in cryo-electron microscopy is one of the main techniques used to determine the structure of biological ensembles at high resolution. Scipion provides the tools to create the whole pipeline to process the information acquired by the microscope and achieve a 3D reconstruction of the biological specimen.

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 ...

Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could easily be adapted for other biological samples. The basic principles for preparing samples, milling, and viewing lamellae are common to all instruments and the protocol can be followed as a general guide to on-grid cryo-lamella preparation for cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET). Electron microscopy grids supporting the cells are plunge-frozen into liquid nitrogen-cooled liquid ethane using a manual or automated plunge freezer, then screened on a light microscope equipped with a cryo-stage. Frozen grids are transferred into a cryo-scanning electron microscope equipped with a focused ion beam (cryoFIB-SEM). Grids are routinely sputter coated prior to milling, which aids dispersal of charge build-up during milling. Alternatively, an e-beam rotary coater can be used to apply a layer of carbon-platinum to the grids, the exact thickness of which can be more precisely controlled. Once inside the cryoFIB-SEM an additional coating of an organoplatinum compound is applied to the surface of the grid via a gas injection system (GIS). This layer protects the front edge of the lamella as it is milled, the integrity of which is critical for achieving uniformly thin lamellae. Regions of interest are identified via SEM and milling is carried out in a step-wise fashion, reducing the current of the ion beam as the lamella reaches electron transparency, in order to avoid excessive heat generation. A grid with multiple lamellae is then transferred to a transmission electron microscope (TEM) under cryogenic conditions for tilt-series acquisition. A robust and contamination-free workflow for lamella preparation is an essential step for downstream techniques, including cellular cryoEM, cryoET, and sub-tomogram averaging. Development of these techniques, especially for lift-out and milling of high-pressure frozen samples, is of high-priority in the field.

Using focused ion beam milling to produce vitreous on-grid lamellae from plunge frozen biological samples for cryo-electron tomography.

使用双光束扫描电子显微镜从玻璃体生物样品制备薄片进行冷冻电子断层扫描

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could ...

Single Particle Cryo-Electron Microscopy: From Sample to Structure

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

Structure determination of macromolecular complexes using cryoEM has become routine for certain classes of proteins and complexes. Here, this pipeline is summarized (sample preparation, screening, data acquisition and processing) and readers are directed towards further detailed resources and variables that may be altered in the case of more challenging specimens.

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 ...

Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires a thin layer of vitreous ice containing the biomolecules of interest. Despite numerous technological advances in recent years that have propelled single-particle cryoEM to the forefront of structural biology, the methods by which specimens are vitrified for high-resolution imaging often remain the rate-limiting step. Although numerous recent efforts have provided means to overcome hurdles frequently encountered during specimen vitrification, including the development of novel sample supports and innovative vitrification instrumentation, the traditional manually operated plunger remains a staple in the cryoEM community due to the low cost to purchase and ease of operation. Here, we provide detailed methods for using a standard, guillotine-style manually operated blot-and-plunge device for the vitrification of biological specimens for high-resolution imaging by single-particle cryoEM. Additionally, commonly encountered issues and troubleshooting recommendations for when a standard preparation fails to yield a suitable specimen are also described.

This manuscript outlines the blot-and-plunge method to manually freeze biological specimens for single-particle cryogenic electron microscopy.

用于单颗粒低温电子显微镜的生物标本的手动印迹和切入式冷冻

Imaging biological specimens with electrons for high-resolution structure determination by single-particle cryogenic electron microscopy (cryoEM) requires ...

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

Recent advances in both instrumentation and image processing software have made single-particle cryo-electron microscopy (cryo-EM) the preferred method for structural biologists to determine high-resolution structures of a wide variety of macromolecules. Multiple software suites are available to new and expert users for image processing and structure calculation, which streamline the same basic workflow: movies acquired by the microscope detectors undergo correction for beam-induced motion and contrast transfer function (CTF) estimation. Next, particle images are selected and extracted from averaged movie frames for iterative 2D and 3D classification, followed by 3D reconstruction, refinement, and validation. Because various software packages employ different algorithms and require varying levels of expertise to operate, the 3D maps they generate often differ in quality and resolution. Thus, users regularly transfer data between a variety of programs for optimal results. This paper provides a guide for users to navigate a workflow across the popular software packages: cryoSPARC v3, RELION-3, and Scipion 3 to obtain a near-atomic resolution structure of the adeno-associated virus (AAV). We first detail an image processing pipeline with cryoSPARC v3, as its efficient algorithms and easy-to-use GUI allow users to quickly arrive at a 3D map. In the next step, we use PyEM and in-house scripts to convert and transfer particle coordinates from the best quality 3D reconstruction obtained in cryoSPARC v3 to RELION-3 and Scipion 3 and recalculate 3D maps. Finally, we outline steps for further refinement and validation of the resultant structures by integrating algorithms from RELION-3 and Scipion 3. In this article, we describe how to effectively utilize three processing platforms to create a single and robust workflow applicable to a variety of data sets for high-resolution structure determination.

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

This article describes how to effectively utilize three cryo-EM processing platforms, i.e., cryoSPARC v3, RELION-3, and Scipion 3, to create a single and robust workflow applicable to a variety of single-particle data sets for high-resolution structure determination.

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

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ever-increasing share of published structural data. Recent advances in TEM technology and automation have boosted both the speed of data collection and quality of acquired images while simultaneously decreasing the required level of expertise for obtaining cryo-EM maps at sub-3 &#197; resolutions. While most of such high-resolution structures have been obtained using state-of-the-art 300 kV cryo-TEM systems, high-resolution structures can be also obtained with 200 kV cryo-TEM systems, especially when equipped with an energy filter. Additionally, automation of microscope alignments and data collection with real-time image quality assessment reduces system complexity and assures optimal microscope settings, resulting in increased yield of high-quality images and overall throughput of data collection. This protocol demonstrates the implementation of recent technological advances and automation features on a 200 kV cryo-transmission electron microscope and shows how to collect data for the reconstruction of 3D maps that are sufficient for de novo atomic model building. We focus on best practices, critical variables, and common issues that must be considered to enable the routine collection of such high-resolution cryo-EM datasets. Particularly the following essential topics are reviewed in detail: i) automation of microscope alignments, ii) selection of suitable areas for data acquisition, iii) optimal optical parameters for high-quality, high-throughput data collection, iv) energy filter tuning for zero-loss imaging, and v) data management and quality assessment. Application of the best practices and improvement of achievable resolution using an energy filter will be demonstrated on the example of apo-ferritin that was reconstructed to 1.6 &#197;, and Thermoplasma acidophilum 20S proteasome reconstructed to 2.1-&#197; resolution using a 200 kV TEM equipped with an energy filter and a direct electron detector.

High-resolution cryo-EM maps of macromolecules can be also achieved by using 200 kV TEM microscopes. This protocol shows the best practices for setting accurate optics alignments, data acquisition schemes, and selection of imaging areas that are all essential for the successful collection of high-resolution datasets using a 200 kV TEM.

使用SPHIRE的电子冷冻显微镜图像的高分辨率单粒子分析

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细菌显示文库的半自动化筛选肽亲和试剂的发现与分离结果分析

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摘要

探索更多视频

此视频中的章节

细菌显示文库的半自动化筛选肽亲和试剂的发现与分离结果分析

β-淀粉脂质诱导纤维蛋白斑点结构异常的光谱分析

单粒子冷冻电子显微镜在结构中测定脂质敏感分通道 trpc3 的表达与纯化

通过低温BN-MS分析进行高分辨率复杂分析

使用沉降机进行冷冻电镜和单颗粒分析

使用双光束扫描电子显微镜从玻璃体生物样品制备薄片进行冷冻电子断层扫描

单颗粒冷冻电子显微镜:从样品到结构

用于单颗粒低温电子显微镜的生物标本的手动印迹和切入式冷冻

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

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

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