Name: using tomoauto: a protocol for high-throughput automated cryo-electron tomography
Uploaded: 2016-01-30T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography at JoVE.com

我们感谢威廉·马戈林医生征求意见。我们感谢来自博士的SerialEM的支持。大卫Mastronarde和陈旭。 DM，BH和JL被授予R01AI087946从国家过敏和传染病研究所，资助R01GM110243和R01GM107629从普通医学科学研究所（NIGMS）和格兰特AU-1714从韦尔奇基金会的支持。直接电子探测器是由美国卫生奖S10OD016279国家机构。

1.小细准备<ol><li>为了使S.菌微细胞，变换1微升质粒pBS58，其组成型表达的大肠杆菌细胞分裂的基因ftsQ，ftsA，和ftsZ超从低拷贝壮观霉素抗性质粒入5微升电感受链霉素抗性5a血清型（M90T钐）细胞通过电穿孔的在2.5千伏为5毫秒，以1毫米试管。 </li><li>商店微细胞的样品在-80℃下在15％甘油在1.5ml微管中的低温。当准备使用时，刮大约5微升用枪头从unthawed微管细胞和悬浮细胞在4毫升的胰蛋白酶大豆肉汤与壮观霉素加入到100微克/毫升的浓度。成长O / N在37℃。 </li><li>吸管2毫升培养从1.2入200ml胰蛋白酶大豆肉汤与壮观霉素再次加入到100微克/毫升的浓度。在37℃下生长至晚对数期。 </li><li>为了丰富小细胞，离心200毫升从1.3的培养物在1000×g离心5分钟。小心倾上清液部分到一个新的离心管中并离心以20,000×g离心10分钟。仔细倒出并丢弃上清液部分，并轻轻混合颗粒使用一个枪头剩余的液体和到1.5ml微量离心管中传输大约100微升沉淀混合物。 </li></ol> 2. EM网格准备<ol><li>放置一个R 2/2多孔碳膜200目铜网上碳朝上载玻片上。 注:R 2/2 200目网格被选择以最大化的倾斜系列可被设置以获得在一个单一的网格，同时仍然支持试样和放置碳膜的边缘，在摄像机的视场的数目在所需的倍率。精细的网格的网格和更小的多孔碳薄膜如R1.2 / 1.3 400目，可用于成像在更高的放大倍率样本;较大的多孔碳薄膜，如R3.5 / 1 200目可使用d表示样品成像在较低的放大率，或如果显微照片不应该包含具有较大的间距，例如R 1的碳边缘和多孔碳薄膜/ 4 200目可以用来帮助进行对准阶段到感兴趣的区域和保护区域从过度曝光在聚焦和跟踪​​例程。 </li><li>放置在辉光放电装置在平台上的幻灯片。 注意:我们使用一个内部装置，其中在阳极和平台已加工到真空干燥器中，由一个高频发生器供电。形成真空后，附加高频发生器探针探针上的阳极和电源1分钟到辉光放电的网格。必要的时间，以辉光放电网格的范围可以从几秒钟到一分钟。改变辉光放电时间可用于诊断问题随试样浓度和出现干燥无玻璃体冰网格。 </li><li>与一组镊子除去网格，并锁定镊子关闭用弹性b和。 </li><li>加入100微升10纳米的胶体金溶液的微量离心管中，在1.4中制备的微细胞，并通过轻弹管用手指混合。随着新的吸管发生4微升在2.2编写电网的混合物。 注:胶体金是有各种尺寸和护理提供应采取的金的尺寸大于5个像素定在所获取的放大要处理的显微照片的像素大小，而不是过大，晦涩特征出于兴趣。 </li><li>准备切入冻结装置;充满液氮的外冷冻容器，然后装满液体乙烷内腔。附加镊子与电网向柱塞杆和柱塞杆锁定到升高位置。 注:用于描述使用商业暴跌冻结设备的协议，见扬库等 [20]。 </li><li>通过仔细触摸一片滤纸到t吸干电网他滴样品直到网格和滤纸分离并在滤纸上的芯吸停止之间的弯液面，然后立即释放柱塞杆，冻结的网格。小心地从柱塞杆除去钳子，并放置在网格到网格支架。 </li><li>用液态氮填充装载区和吸收泵容器准备冷冻电镜中转站。一旦装载区域是在液氮温度下发生的网格支架，并在装载区在显微镜标本盒。 </li><li>小心地取出锁环，这是早期的波拉显微镜或以后的型号C风格的夹环或者是小螺纹锁环;使用镊子将EM电网进入墨盒，然后轻轻地重新安装锁环放回盒固定网格。 </li><li>从显微镜中取出多个样品支架，并将其安装到中转站。将试样墨盒插入墨盒使用的镊子持有人Ð缩回从装载区域中的保持器和多个试样保持器传回的显微镜。 注:参见陈等人 21为可视的协议细节2.1-2.9。 </li></ol> 3.高通量自动化倾斜系列集合<ol><li>集低倍率地图的<ol><li>通过点击"打开"的SerialEM 10的"导航"菜单中打开一个新的浏览器窗口（http://bio3d.colorado.edu/SerialEM） </li><li>查找包含可接受的成像条件方格（ 即薄冰，没有污染，感兴趣的主题）使用荧光屏幕在低倍率（〜2,300X为小细胞标本）。 注:[可选:该步骤可以与SerialEM通过剪接整个电网自动化，但它能够更快简单地手动选择少数地区] </li><li>调整阶段eucentric高度倾斜样品架至50°再调整的z高度，直到该级的XY平移是最小的倾斜和非倾斜视图之间。 </li><li>移动到方格的中心，点击Navigator窗口中的"添加阶段名次"按钮来保存当前舞台上的地位。 </li><li>继续步骤3.1.1-4以上，直到所有接受方格级职位已被保存。 </li><li>通过单击"文件"菜单中的"新建蒙太奇"打开一个新的蒙太奇MRC文件。在蒙太奇设置对话框打开后，选择一个数字X和Y枚将收购整个方格（ 例如，10×10的标准的200网格）的。使用高分级，如8，选择"移动相反的图像转移阶段"和"跳过用于对齐件相关"单选按钮。 </li><li>在浏览器窗口中单击第一个舞台上的地位，并把它设置为通过检查"采集"复选框被收购。在浏览器窗口中的每个阶段的位置重复此。 </li><li>打开浏览器获取对话框通过在"导航"菜单中单击"获取的积分"。选中"采集地图图像"和"粗eucentricity"复选框，并确保所有其他复选框都未选中。点击"继续"，收集了蒙太奇在每个阶段的位置。 </li></ol></li><li>倾斜系列采集<ol><li>在浏览器窗口中选择被收购的地图之一，并点击"载入地图"按钮。 </li><li>在浏览器窗口中，单击"添加点"按钮，然后选择点在其中获得倾斜系列地图。然后点击"停止添加点"按钮。重复每个收集的地图。 </li><li>在相机菜单中选择"参数"，并为重点，审判和记录模式定义的参数。 [可选:剂量分级的数据可以为拍摄模式下的参数指定] </li><li>选择Navigator窗口中的一个点，并检查"倾斜系列9;复选框。在打开选择参数倾翻式系列设置对话窗口所需的倾斜系列采集。重复在导航窗口中选定点的休息，但不要选择地图。 </li><li>在导航菜单中再次选择"获取的积分"。在导航器获取对话框中选择"重新调整项"，自动对焦"和"粗eucentricity"的初步任务，并选择"获取倾斜系列"作为首要任务，并选择"关闭柱阀，在结束"当所有关闭列点的已收集。一旦继续倾斜系列将在每个地图每个点采集。 </li></ol></li></ol> 4.高通量自动化倾斜系列加工和重建使用Tomoauto <ol><li>梁诱发运动的剂量分次数据修正[可选] 注:Tomoauto使用MOTIONCORR 22（http://cryoem.ucsf.edu/software/driftcorr.ht毫升），从剂量分次取出显微光束引起的运动。 NOTIONCORR 16必须安装在系统上。 <ol><li>与原来的倾斜系列，从SerialEM 10在当前工作目录输出日志和个人剂量分级的所有图像，在终端执行以下命令: dose_fractioned_to_stack &lt;filename.st&gt; &lt;filename.st&gt;是倾斜系列以处理的名称。 </li></ol></li><li>对齐的倾斜系列和重建 注:Tomoauto默认使用IMOD 13（http://bio3d.colorado.edu/imod/）来处理倾斜系列自动基准模型生成，比对，确定对比度传递函数（CTF），CTF校正23，和重建。另外用户在tomoauto选择使用RAPTOR 24（包含在IMOD）的自动化基准模型生成，周大福FIND4 25（http://grigoriefflab.janelia.org/ctf）来确定CTF，和tomo3d 26（https://sites.google.com/site/3demimageprocessing/tomo3d），用于重建或以软件产品的任何组合组态。此每个包中可用的配置以及参数是由可以被编辑以适合在实验室中最常用的值，同时本地配置文件还可以创建用于特定样本细节的参数，收集一个全局配置文件处理设置或个人倾斜系列。那些希望使用所有包装必须安装在系统上。 <ol><li>随着倾斜系列在当前的工作目录，在终端执行命令 tomoauto --CTF --mode =对齐&lt;filename.st&gt; &lt;fid_diam&gt; &lt;filename.st&gt;是倾斜系列进行处理并&lt;fid_diam&gt;是diame之三的以纳米为单位的基准标记。此命令将调整和自动估计倾斜系列的CTF。有可能跳过CTF处理通过从命令中--CTF选项。 </li><li>检查对准倾斜一系列可视在对准处理任何明显的误差和通过执行的命令检查所估计的CTF: 3dmod &lt;文件名​​&gt; .ali submfg &lt;文件名​​&gt; _ctfplotter.com 分别为， 其中 &lt;文件名​​&gt;是倾斜系列无后缀的名称。同时检查tomoauto命令的输出看到的一致性，这是调整的质量进行定量统计生成的平均剩余误差。 </li><li>给定一个可接受的定位通过执行命令进行处理: tomoau以--CTF --mode =重构&lt;filename.st&gt; &lt;fid_diam&gt; 使用相同的用户替换为4.2.1。此命令将纠正周大福，从倾斜系列擦除基准标记和计算的重建。再次CTF处理可以跳过在4.2.1。 </li><li> [可选]要跳过目视检查步骤，并全面实现自动化处理与重建执行命令 tomoauto --CTF &lt;filename.st&gt; &lt;fid_diam&gt; </li><li> [可选]要使用特定的本地配置，请参阅如何生成一个本地配置文件tomoauto文件，然后执行命令 tomoauto [选项] -L &lt;local_config&gt; &lt;filename.st&gt; &lt;fid_diam&gt; 其中&lt;local_config&gt;是本地的conf名iguration文件。 </li></ol></li></ol> 5.子断层扫描平均注:我们使用的i3包15（http://www.electrontomography.org/）来处理子断层扫描平均的实验，但协议中所述，一般适用于大多数可用的子断层扫描平均软件packages16to处理子断层扫描平均的实验，但协议中所述，一般适用于大多数可用的子断层扫描平均软件包16-18。 <ol><li>随着当前工作目录重建的断层图像开拓断层扫描粒子采摘通过执行以下命令: tomopick &lt;文件名&gt; .REC 其中&lt;文件名&gt;是在4.2.2。在打开使用鼠标左键点击第一个基础体温，然后针尖选择一个injectisome并使用向上和向下箭头键来即兴通过tomogra的切片窗口米选择这种方式的所有可见injectisomes。这存储在文本文件中定义的结构的长轴以及推定的两个3欧拉角描述结构的取向的坐标。 </li><li>计算提取从通过执行命令集中在定义的长轴的中点的断层图像400 3体素立方体: 夹大小调整-cx &lt;X&gt; -cy &lt;Y&gt; -cz &lt;Z&gt; -ix 400 -Iy 400 -Iz 400 &lt;文件名&gt; .REC &lt;文件名&gt; _001.mrc 其中&lt;x&gt;，&lt;y&gt;和&lt;Z&gt;是结构的中点坐标和&lt;文件名&gt;是在4.2.2。所提取的立方体的尺寸应该由结构和所用的放大倍数而变化，并且应该足够大，以适当地包围感兴趣的结构，这对于该样品是400 3体素。 </li><li>下采样（斌）子断层扫描由四个因素通过执行以下命令，以减少计算时间初始校准: binvol -b 4 &lt;文件名&gt; _001.mrc &lt;文件​​名&gt; _001.bin4.mrc 其中&lt;文件名&gt;是在5.2。 </li><li>应用决定欧拉角子断层和计算的全球平均通过执行以下命令来产生初始模板。 I3totsum.sh </li><li>对准并使用胞质区域的二元分类掩模分类向下抽样子断层图像。在傅立叶空间中进行的子断层图像平均化，以尽量减少缺失槽楔工件断层扫描的特性。对于分档4的数据，请使用SAMPFACT ="4 4 4"。 i3mramsacls.sh </li><li>重复步骤5.5采用子断层图像下采样由两个（SAMPFACT =＆一个因子＃34; 2 2 2"），并再次与原始数据（SAMPFACT ="1 1 1"）。 i3mramsacls.sh </li></ol>

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V., 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=Electron+cryotomography+sample+preparation+using+the+Vitrobot.">Electron cryotomography sample preparation using the Vitrobot.</a> Nat. Protoc. 1 (6), 2813-2819 (2007).</li><li>Chen, S., 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=Electron+Cryoelectrontomography+of+Bacterial+Cells.">Electron Cryoelectrontomography of Bacterial Cells.</a> J. Vis. Exp. (39), e1943 (2010).</li><li>Li, X., 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=Electron+counting+and+beam-induced+motion+correction+enable+near-atomic-resolution+single-particle+cryo-EM.">Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM.</a> Nat. Methods. 10 (6), 584-590 (2013).</li><li>Xiong, Q., Morphew, M. K., Schwartz, C. L., Hoenger, A. H., Mastronarde, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CTF+determination+and+correction+for+low+dose+tomographic+tilt+series.">CTF determination and correction for low dose tomographic tilt series.</a> J. Struct. Biol. 168 (3), 378-387 (2009).</li><li>Amat, F., Moussavi, F., Comolli, L. R., Elidan, G., Downing, K. H., Horowitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markov+random+field+based+automatic+image+alignment+for+electron+tomography.">Markov random field based automatic image alignment for electron tomography.</a> J. Struct. Biol. 161 (3), 260-275 (2008).</li><li>Rouhou, A., Grigorieff, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CTFFIND4:+Fast+and+accurate+defocus+estimation+from+electron+micrographs.">CTFFIND4: Fast and accurate defocus estimation from electron micrographs.</a> bioRxiv. , (2015).</li><li>Agulleiro, J. I., Fernandez, 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=Tomo3D+2.0+&#8211;+Exploitation+of+Advanced+Vector+eXtensions+(AVX)+for+3D+reconstruction.">Tomo3D 2.0 &#8211; Exploitation of Advanced Vector eXtensions (AVX) for 3D reconstruction.</a> J. Struct. Biol. 189 (2), 147-152 (2015).</li><li>Zhao, X., Zhang, K., Boquoi, T., Hu, B., Motaleb, M. A., Miller, K., James, M., Charon, N. W., Manon, M. D., Norris, S. J., Li, C., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-Electron+Tomography+Reveals+the+Sequential+Assembly+of+Bacterial+Flagella+in+Borrelia+burgdorferi.">Cryo-Electron Tomography Reveals the Sequential Assembly of Bacterial Flagella in Borrelia burgdorferi.</a> Proc Natl Acad Sci U S A. 110 (35), 14390-14395 (2013).</li><li>Hu, B., Margolin, W., Molineux, I. J., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bacteriophage+T7+Virion+Undergoes+Extensive+Structural+Remodeling+during+infection.">The Bacteriophage T7 Virion Undergoes Extensive Structural Remodeling during infection.</a> Science. 339 (6119), 576-579 (2013).</li><li>Liu, J., Hu, B., Morado, D. R., Jani, S., Manson, M. D., Margolin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=W:+Molecular+architecture+of+chemoreceptor+arrays+revealed+by+cryoelectron+tomography+of+Escherichia+coli+minicells.">W: Molecular architecture of chemoreceptor arrays revealed by cryoelectron tomography of Escherichia coli minicells.</a> Proc Natl Acad Sci USA. 109 (23), e1481-e1488 (2012).</li><li>Russo, C. J., Passmore, L. 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作者宣称，他们有没有竞争的财务权益。

此处所描述的高通量方法，使我们能够处理1917低温倾斜系列和产生超过4500分断层图像的完好S的菌 injectisome 19。所收集的数据导致原位 injectisome的详细的表征，包括胞质排序复杂。该方法也被用来可视化几个突变的细胞与特异性缺失推定的蛋白质组分，这有助于阐明injectisome的排序平台的组成。我们的方法提供了新的途径，调查injectisome的结构与功能的关系。其结果是?...

III型分泌系统（T3SS）是必不可少的毒力决定于许多革兰氏阴性病原体。的injectisome，也被称为针复杂，是所需的效应蛋白直接易位从细菌到真核宿主细胞1,2中央T3SS机。所述injectisome包括细胞外针，一个基体，和胞质复杂也是已知作为拣杂3。先前的研究已阐明了从沙门氏菌和 志贺氏菌纯化的injectisomes的3-D结构，随着主要基体蛋白4,5的原子结构。最近在从沙门氏菌， 志贺氏菌和耶尔森氏菌 injectisomes 原位结构进行了揭示的低温的ET 6 7，然而，胞质复杂，效应子选择和针组件必需的，还没有被可视化这些结构。 低温-ET是MOSŤ合适技术其天然细胞环境（原位）内成像分子机器在纳米级分辨率。尽管如此，通过Cryo-ET可达到的分辨率由样品厚度的限制。为了克服缺点，我们在毒痢疾杆菌菌株进行基因改造，以产生小细胞的冷冻ET足够薄成像完好injectisomes。冷冻的ET的另一个限制是样品诱导的电子束，它非常快速地破坏在样品中的高分辨率信息的辐射的敏感性。其结果是，非常低剂量用于个体倾斜图像，使合适的剂量可以分布在全倾斜系列。这大大降低了在最终的重建的信噪比（SNR），这使得难以区分的大量的断层图像噪声的被检体的结构特征和限制了可以由永冻达到的分辨率ET。 Conventi可用于Onal地区的图像处理，如傅立叶和现实空间滤波器以及向下采样，以增加对比度，但在过滤掉大部分的高分辨率信息的费用。近来，子断层图像平均化使得有可能大大提高了信噪比并随后在某些情况下，以亚纳米水平8,9的尾声。配合物的更详细的分析成为可能通过计算提取数千含子断层图像从原来的断层图像，然后对准并平均所述子断层图像感兴趣的区域，以确定在具有较高SNR和更高分辨率的原位复杂的结构。这些方法可以用遗传学方法，以提供更大的见解大分子组装，并在天然细胞环境的动态构象进行集成。 在一般情况下，几十甚至几十万子断层需要，以确定高被平均-分辨率结构原位 。采集的倾斜系列足够数量需要产生这种大数目的子断层图像很快成为一个瓶颈。所得倾斜系列往往受光束诱发移位，阶段齿隙，以及倍率，旋转和歪斜缺陷，这必须得到解决，以使倾斜系列对准前重建。倾斜系列典型地通过跟踪金基准标记，这是传统选过倾斜系列的检查手动，引起另一个瓶颈对齐。许多软件包已通过计算机控制的电子显微镜10，11，12，倾斜系列对准和重建13，14和子断层图像平均15-18开发用于自动倾斜系列采集。由于这些产品的处理在低温的ET的工作流分立操作，就变成希望建立一个更高的抽象水平进入过程的Systema角度讲简化整个计划到一个单一的管道。因此，我们开发了软件包装库"tomoauto"旨在组织一些这些包成一个单一的半自动化单元，允许简单的用户操作，同时保持以集中的方式各成分的完整配置。该库是开源的，有据可查的，不断发展和可以自由使用，定制开发或进一步集成通过网上远程源代码库（http://github.com/DustinMorado/tomoauto）的手段。 这种高通量冷冻ET管道已被用来可视化完好injectisomes S中菌的微细胞。使用该方法生成总共1917断层图像，揭示一个高分辨率在完整的机器的原位结构，包括由子断层图像均 19确定的细胞质分拣平台。连同野生型和米的分子模拟utant机，我们的高通量管道提供了一种新的途径，以了解在天然细胞环境的完整injectisome的结构和功能。

<table><tbody><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G9012</td><td></td></tr><tr><td>Tyrptic Soy Broth</td><td>Sigma-Aldrich</td><td>22092</td><td></td></tr><tr><td>Spectinomycin</td><td>Sigma-Aldrich</td><td>S0692</td><td></td></tr><tr><td>Electroporation Apparatus</td><td>Bio-rad</td><td>165-2100</td><td></td></tr><tr><td>1 mm Cuvette</td><td>BTX</td><td>45-0124</td><td></td></tr><tr><td>1.5 ml Cryogenic Tube</td><td>Thermoscientific</td><td>5000-1020</td><td></td></tr><tr><td>1.5 ml Microcentrifuge Tube</td><td>Sigma-Aldrich</td><td>Z336769</td><td></td></tr><tr><td>Holey Carbon Grids</td><td>Quantifoil&lt;br/&gt; (Electron Microscopy Sciences)</td><td>Q2100CR2</td><td>R2/2 200 Cu</td></tr><tr><td>Glow Discharge Device</td><td>In-House</td><td></td><td>Commercial Alternative Available</td></tr><tr><td>Vacuum Desiccator</td><td>Sigma-Aldrich</td><td>Z119016&amp;nbsp;</td><td>Used in In-House Glow Discharge Device</td></tr><tr><td>High-Frequency Generator</td><td>Electro-Technic Products</td><td>BD-10A</td><td>Used in In-House Glow Discharge Device.&amp;nbsp; CAUTION: This device generates high voltages.</td></tr><tr><td>Centrifuge</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td>Dumont&lt;br/&gt; (Electron Microscopy Sciences)</td><td>72705-D</td><td>Style 5 Anti-magnetic</td></tr><tr><td>Colliodal Gold</td><td>Aurion</td><td></td><td>BSA 10nm</td></tr><tr><td>Filter Paper</td><td>Whatman</td><td></td><td>#2</td></tr><tr><td>Ethane&amp;nbsp;</td><td>Matheson Tri-Gas</td><td>UN1035</td><td></td></tr><tr><td>Nitrogen</td><td>Matheson Tri-Gas</td><td>UN1977</td><td></td></tr><tr><td>Plunger Device</td><td>In-House</td><td></td><td>Commercial Alternative Available</td></tr><tr><td>Cryogenic Grid Storage Box</td><td>Electron Microscopy Sciences</td><td>71166-30</td><td></td></tr><tr><td>Transmission Electron Microscope</td><td>FEI</td><td></td><td>Tecnai Polara F30&lt;br/&gt; (300 KeV)</td></tr><tr><td>Direct Detection Device Camera</td><td>Gatan</td><td></td><td>K2 Summit</td></tr><tr><td>Tomogram Acquisiton Software</td><td>SerialEM</td><td></td><td>http://bio3d.colorado.eud/SerialEM Alternatives: UCSF Tomography, Leginon, FEI Batch Tomography</td></tr><tr><td>Beam-induced Motion Correction Software</td><td>MOTIONCORR</td><td></td><td>http://cryoem.ucsf.edu/software/driftcorr.html Requires &gt;2GB Nvidia GPU</td></tr><tr><td>Tilt-Series Alignment Software</td><td>IMOD</td><td></td><td>http://bio3d.colorado.edu/IMOD Alternatives: XMIPP, Protomo</td></tr><tr><td>Automatic Fiducial Marker Modelling Software</td><td>IMOD</td><td></td><td>Alternatives: RAPTOR (Included in IMOD0&lt;br/&gt; (Usable in tomoauto)</td></tr><tr><td>CTF Determination Software</td><td>IMOD</td><td></td><td>Alternatives: CTFFIND http://grigoriefflab.janelia.org/ctf&lt;br/&gt; (Usable in tomoauto)</td></tr><tr><td>Tilt-Series Reconstruction Software</td><td>tomo3d</td><td></td><td>https://sites.google.com/site/3demimageprocessing/tomo3d Alternatives: IMOD, XMIPP http://xmipp.cnb.csic.es , Protomo</td></tr><tr><td>Tilt-Series Automated Processing Software</td><td>tomoauto</td><td></td><td>https://github.com/DustinMorado/tomoauto</td></tr><tr><td>Particle Picking Software</td><td>i3</td><td></td><td>http://www.electrontomography.org Alternatives: IMOD</td></tr><tr><td>Subvolume Averaging Software</td><td>i3</td><td></td><td>Alternatives: PEET http://bio3d.colorado.edu/PEET, Dynamo https://dynamo.bioz.unibas.ch , PyTom http://pytom.org</td></tr></tbody></table>

using tomoauto: a protocol for high-throughput automated cryo-electron tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

小细胞S.样品菌 ，收集并处理的结果显示在原理图1采用tomoauto在图2中详述的管道下面。 使用SerialEM 10，其允许高通量倾斜系列采集在上低倍率蒙太奇地图由用户指定的点（ 图3）倾斜系列收集。使用剂量分馏模式直接检测器件照相机上，以减少束诱发运动22（图4），收集显微镜?...

Watch this Scientific Journal Video about Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography at JoVE.com

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

我们提出如何利用高通量低温电子断层扫描，以确定高分辨率的分子机器的结构原位的协议。该协议允许大量数据要处理，避免了常见的瓶颈，并减少停机时间资源，从而允许用户集中在重要的生物的问题。

使用Tomoauto:一个协议，用于高通量自动化低温电子断层扫描

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

using-tomoauto-protocol-for-high-throughput-automated-cryo-electron

Research

JoVE Journal

Bioengineering

10.9K 次观看.  University of Texas Health Science Center at Houston. 我们提出如何利用高通量低温电子断层扫描，以确定高分辨率的分子机器的结构原位的协议。该协议允许大量数据要处理，避免了常见的瓶颈，并减少停机时间资源，从而允许用户集中在重要的生物的问题。 

视频: Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

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

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

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

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

生物工程

Electron Cryotomography of Bacterial Cells

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bridge the resolution gap between fluorescence light microscopy and higher-resolution methods such as X-ray crystallography and NMR spectroscopy.
Electron cryotomography (ECT) is an emerging technology that does just this, allowing the ultrastructure of cells to be visualized in a near-native state, in three dimensions (3D), with "macromolecular" resolution (~4nm).1, 2 In ECT, cells are imaged in a vitreous, "frozen-hydrated" state in a cryo transmission electron microscope (cryoTEM) at low temperature (&lt; -180&deg;C). For slender cells (up to ~500 nm in thickness3), intact cells are plunge-frozen within media across EM grids in cryogens such as ethane or ethane/propane mixtures. Thicker cells and biofilms can also be imaged in a vitreous state by first "high-pressure freezing" and then, "cryo-sectioning" them. A series of two-dimensional projection images are then collected through the sample as it is incrementally tilted along one or two axes. A three-dimensional reconstruction, or "tomogram" can then be calculated from the images. While ECT requires expensive instrumentation, in recent years, it has been used in a few labs to reveal the structures of various external appendages, the structures of different cell envelopes, the positions and structures of cytoskeletal filaments, and the locations and architectures of large macromolecular assemblies such as flagellar motors, internal compartments and chemoreceptor arrays.1, 2
In this video article we illustrate how to image cells with ECT, including the processes of sample preparation, data collection, tomogram reconstruction, and interpretation of the results through segmentation and in some cases correlation with light microscopy.

We illustrate here how to use electron cryotomography (ECT) to study the ultrastructure of bacterial cells in near-native states, to "macromolecular" (~4 nm) resolution.

细菌细胞的电子Cryotomography

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for ...

生物学

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 ...

生物化学

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

In the past several years, technological and methodological advancements in single-particle cryo-electron microscopy (cryo-EM) have paved a new avenue for the high-resolution structure determination of biological macromolecules. Despite the remarkable advances in cryo-EM, there is still scope for improvement in various aspects of the single-particle analysis workflow. Single-particle analysis demands a suitable software package for high-throughput automatic data acquisition. Several automatic data acquisition software packages were developed for automatic imaging for single-particle cryo-EM in the last eight years. This paper presents an application of a fully automated image acquisition pipeline for vitrified biomolecules under low-dose conditions. 
It demonstrates a software package, which can collect cryo-EM data fully, automatically, and precisely. Additionally, various microscopic parameters are easily controlled by this software package. This protocol demonstrates the potential of this software package in automated imaging of the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) spike protein with a 200 keV cryo-electron microscope equipped with a direct electron detector (DED). Around 3,000 cryo-EM movie images were acquired in a single session (48 h) of data collection, yielding an atomic-resolution structure of the spike protein of SARS-CoV-2. Furthermore, this structural study indicates that the spike protein adopts two major conformations, 1-RBD (receptor-binding domain) up open and all RBD down closed conformations.

Single-particle cryo-electron microscopy demands a suitable software package and user-friendly pipeline for high-throughput automatic data acquisition. Here, we present the application of a fully automated image acquisition software package, Latitude-S, and a practical pipeline for data collection of vitrified biomolecules under low-dose conditions.

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 ...

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to occur in some cyanobacteria before cell division. However, due to the large cell size and the transient character, it is difficult to investigate the structure in detail. To overcome the difficulties, first, DNA compaction is reproducibly produced in the cyanobacterium Synechococcus elongatus PCC 7942 by synchronous culture using 12 h each light/dark cycle. Second, DNA compaction is monitored by fluorescence microscopy and captured by rapid freezing. Third, the detailed structure of DNA compacted cells is visualized in three dimensions (3D) by high voltage cryo-electron tomography. This set of methods is widely applicable to investigate transient structures in bacteria, e.g. cell division, chromosome segregation, phage infection etc., which are monitored by fluorescence microscopy and directly visualized by cryo-electron tomography at appropriate time points.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. Synchronous cultivation, monitoring by fluorescence microscopy, rapid freezing, and high voltage cryo-electron tomography are used. A protocol for these methodologies is presented, and future applications and developments are discussed.

高压低温电子层析成像对蓝藻 DNA 压实的可视化研究

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.

优化低温电子断层扫描数据采集策略

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current ...

Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, replication, segregation, etc.) remain poorly understood, partially due to the lack of experimental approaches to high-resolution visualization of specific chromatin loci in structurally preserved nuclei. Here we present a protocol for the visualization of replicative domains in monolayer cell culture in situ,&#160;by combining EdU labeling of newly synthesized DNA with subsequent label detection with Ag-amplification of Nanogold particles and ChromEM staining of chromatin. This protocol allows for the high-contrast, high-efficiency pre-embedding labeling, compatible with traditional glutaraldehyde fixation that provides the best structural preservation of chromatin for room-temperature sample processing. Another advantage of pre-embedding labeling is the possibility to pre-select cells of interest for sectioning. This is especially important for the analysis of heterogeneous cell populations, as well as compatibility with electron tomography approaches to high-resolution 3D analysis of chromatin organization at sites of replication, and the analysis of post-replicative chromatin rearrangement and sister chromatid segregation in the interphase.

This protocol presents a technique for high-resolution mapping of replication sites in structurally preserved chromatin in situ that employs a combination of pre-embedding EdU-streptavidin-Nanogold labeling and ChromEMT.

通过电子断层扫描对超构造保存的染色质中的复制结构域进行成像

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, ...

Cryo-Electron Tomography Remote Data Collection and Subtomogram Averaging

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved automated acquisition strategies, preparative techniques that expand the possibilities of what the electron microscope can image at high-resolution using cryo-ET and new subtomogram averaging software. Additionally, data acquisition has become increasingly streamlined, making it more accessible to many users. The SARS-CoV-2 pandemic has further accelerated remote cryo-electron microscopy (cryo-EM) data collection, especially for single-particle cryo-EM, in many facilities globally, providing uninterrupted user access to state-of-the-art instruments during the pandemic. With the recent advances in Tomo5 (software for 3D electron tomography), remote cryo-ET data collection has become robust and easy to handle from anywhere in the world. This article aims to provide a detailed walk-through, starting from the data collection setup in the tomography software for the process of a (remote) cryo-ET data collection session with detailed troubleshooting. The (remote) data collection protocol is further complemented with the workflow for structure determination at near-atomic resolution by subtomogram averaging with emClarity, using apoferritin as an example.

The present protocol describes high-resolution cryo-electron tomography remote data acquisition using Tomo5 and subsequent data processing and subtomogram averaging using emClarity. Apoferritin is used as an example to illustrate detailed step-by-step processes to achieve a cryo-ET structure at 2.86 &#197; resolution.

冷冻电子断层扫描远程数据收集和断层扫描平均

Cryo-electron tomography (cryo-ET) has been gaining momentum in recent years, especially since the introduction of direct electron detectors, improved ...