This work was supported by the Academy of Finland (130750 and 218080 to J.T.H.), the Wellcome Trust (090532/Z/09/Z; 089026/Z/09/Z to T.A.B.), and by the MRC (MR/J007897/1 to J.T.H and T.A.B; MR/L009528/1 to T.A.B.).

为计算路线和病毒糖蛋白的尖峰随后均得到详细的协议概述。协议如下图1中所示的工作流程，并结合一种自动化搜索使用初始模板结构和黄金标准结构细化的尖峰。 输入数据，该协议是一组病毒体的断层重建的。一个断层图像中包含的一个或多个病毒粒子。最初，尖峰的一小部分是手工采摘并用来平均水平，完善两个独立的模型。这些模型用于自动定位尖峰上的所有病毒颗粒。最后，两个独立的精炼进行运行，将所得的平均值进行比较，并结合以产生最终的结构。 细化方法证明了使用程序从Jsubtomo包。从Bsoft包28程序用于一般图像Processing任务和分子图形包UCSF嵌合体29是用于可视化的结果。个别课程以斜体字和文件格式，给出的名称标注有大写的文件扩展名。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig1highres.jpg" width="600" /> 图1:用于确定从形性膜病毒糖蛋白刺突复合物的结构总体战略中的数字对应的协议在不同的章节<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51714/51714fig1highres.jpg" target="_blank">，请点击这里查看该图的放大版本。</a> 1，从全尺寸断层提取病毒分卷<ol><li>在X线断层手动拾取的病毒颗粒。 <ol><li>打开bshow断层图像文件。定义一个病毒partic的中心坐标使用乐颗粒采摘工具。重复此步骤，直到所有的病毒粒子都被处理。保存病毒坐标在星形文件。 </li><li>直到所有的断层图像进行了处理，重复步骤1.1.1。请记下病毒粒子直径的像素。 注:如果尖峰很难看到断层，使用bfilter到低通滤波器的断层图像，以80的分辨率（参数"-bandpass 80,10000"）。 </li></ol></li><li>运行jsubtomo.py中提取模式（参数"--mode提取物"）中提取病毒分卷到单个卷中的文件。使用保存在步骤1.1.1输入文件STAR文件。 <ol><li>给一大小（参数"--size"）〜比在数据集的最大病毒体大25％。 例如 ，如果病毒是〜200个像素的直径，使用尺寸250×250×250像素。 </li><li>病毒粒子体积的输出文件将在MAP格式。此外，创建一个伴随STAR文件为每个病毒粒子的体积（parame之三"--output"）。 </li></ol></li><li>正常化病毒卷bimg（参数"-rescale 0,1"）。 </li></ol> 2，生成两个独立的初始模型<ol><li>匹克在病毒分卷尖峰的一个子集。 <ol><li>打开bshow病毒体分卷映射文件。定义使用粒子采摘工具，通过工具箱窗口访问穗的中心坐标。重复此步骤，直到所有分明尖峰进行了处理。保存秒杀坐标在星文件。 </li><li>如有必要，重复步骤2.1.1其他病毒体，直到大约200糖蛋白尖刺已被处理。注意像素秒杀尺寸。 注:〜200尖峰是一个粗略的指引，更可能需要的一些应用。如果可能的话，目的是挑选尖峰在不同的方位。避免只采摘俯视图。 </li></ol></li><li>通过运行jviews.p分配一个初始视图向量的尖峰年。使用步骤2.1.1输入文件生成的STAR文件。 注:此图矢量近似于参照病毒的刺突的方向。 <ol><li>使用中央协调分配意见的球形病毒颗粒。例如，如果病毒体被集中（步骤1.2之后）在一个盒子具有250×250×250个像素的大小，中心坐标是125125125像素（选项"--Radial 125125125"）。 </li><li>要指定一个丝状病毒的看法，通过使用在步骤2.1.1作为输入文件中定义的文件之星打开bshow每个病毒分卷。限定使用灯丝拾取工具灯丝的两端点，并保存STAR文件。重复此步骤，直到所有明星的文件已被更新并运行jviews.py使用更新之星文件作为输入文件（--option段）。 </li></ol></li><li>使用jsubtomo_create_mask.py产生真正的空间屏蔽。确保在实际空间掩模是同一三维地球的离子作为将被用于平均的结构（参见2.6.2），并足够大以包含两个尖峰和底层膜的贴剂。 </li><li>使用jsubtomo_create_wedgemask.py产生倒易空间的面具。倒数空间掩码用于排除的区域中的"缺失槽楔&#39;从单一轴断层数据收集所得的区域。确保它是相同的尺寸，为将用于所述平均结构（见2.6.2）。 </li><li>生成一个选择文件（SEL文件）限定属于设定"1"和"2"使用jsubtomo_evenodd.py并在步骤2.2中产生的STAR文件的病毒粒子。 </li><li>使用jsubtomo_create_averages.py产生两个初始平均值。 <ol><li>使用步骤2.5作为输入文件生成的SEL文件。 </li><li>举一个大小（参数"--size"）至少32像素比秒杀的最大尺寸。 例如 ，如果秒杀〜40个像素长，使用尺寸72×72×72像素。 </li><li>适用于高对称性（ 如 C100）的平均值（参数"--symmetry C100"），近似围绕秒杀的长轴圆柱形的平均水平。使用对称，以减少在初始平均噪声。 </li><li>项目（参数"--suffix"）的输出文件提供一个唯一的名称。 </li><li>使用在步骤2.3中产生的真实空间掩模掩盖远背景（参数"--mask"）。使用在步骤2.4中产生的倒易空间掩模占信号由丢失楔（参数"--mask"）的存在下引入的损耗。 </li></ol></li></ol> 3，黄金标准迭代对齐和两个初始穗模型的平均反复调整和平均在第2与jsubtomo_iterate_gold.py生成的两个初始模型。 <ol><li>阶段一，这个阶段的目的是改进观察向量的方向。输入文件是在步骤2.5生成的SEL文件。 <ol><li>使用一个8度角取样（参数"--angles 8,8,8"）。 </li><li>允许在穗视图向量的方向的16度的变化（参数"--thetaphilimit 16"），但保持周围的穗长轴线固定的（参数"--alphalimit 0"）的角度。 </li><li>让小平移的变化（ 例如 ，5像素）来解释不准确的尖峰人工采摘（参数"--shiftlimit 5"）。 </li><li>适用于高对称性（ 如 C100）的平均值（参数"--symmetry C100"），近似围绕秒杀的长轴圆柱形的平均水平。 注:对称化是用来减少在平均噪声。 </li><li>使用真正的空间屏蔽和步骤2.3和2.4生成的倒易空间面具来掩盖周围走尖峰和账户失踪的楔形（参数"--M问"和"--mask"，分别）。 </li><li>使用在2.6生成的两个MAP文件（由标记"偶"表示，并在该文件名"奇"）作为模板（参数"--Template1"和"--Template2"，分别）。 </li><li>应用低通滤波器50的分辨率（参数"--resolution"），以防止对准偏差。 </li><li>应用2的仓​​因素，加快细化（参数"--bin 2"）。 </li><li>运行5次迭代调整，平均的（参数"--firstiter 1 --lastiter 5"）。 </li></ol></li><li>第二阶段。这个阶段的目的是缩小视图四周矢量的角度。输入文件是步骤3.1.9输出SEL文件。 <ol><li>通过打开在步骤3.1.9最后一次迭代中产生的低通滤波的MAP文件测量尖峰的精确尺寸在bshow（由变量"_lp"中的文件名 ​​表示）。产生一个新的现实空间屏蔽类似于步骤2.3定义秒杀，但不包括大部分的膜和邻近的尖峰。 注:面罩的最佳尺寸取决于大小和尖峰的特征。 </li><li>用8度角取样（参数"--angles 8,8,8"）作为之前。 </li><li>允许在围绕鞋钉视图向量（参数" - alphalimit 180"）的角180度的变化而保持的θ和披角度固定（参数"--thetaphilimit 0"）。 </li><li>不要让任何翻译班（参数"--shiftlimit 0"）。 </li><li>不适用的平均值（省略参数"--symmetry"）的任何对称性。 </li><li>使用在步骤2.4中产生的倒易空间掩模占缺失槽楔。 </li><li>使用步骤3.1.9的最后一次迭代而不对称性所产生的两个Map文件作为模板（参数R（按标签"even_nosym"，并在文件名"odd_nosym"表示）20; - template1的"和"--Template2"，分别）。 </li><li>在第一次迭代应用低通滤波器50的分辨率（参数"--resolution"），以防止对准偏差。两个独立的平均值之间自动调整，在后续的迭代滤波器参数的基础上的黄金标准傅立叶壳牌相关（FSC）（参数"--adaptivefilter"）。用0.143，标准（参数"--fsccrit 0.143"）。不允许细化过去的对比度传递函数（CTF）（参数"--minhires"）的第一个零，除非CTF校正已被施加到X线断层图。 </li><li>运行5-10重复排列和平均（ 例如 ，参数"--firstiter 6 --lastiter 15"）。监视中的变化报道的分辨率。当没有显著变化观察到的，该过程可以停止。 </li></ol></li><li>评估对称度由examini结构纳克在嵌合体 。 例如所得的低通滤波后的MAP文件（文件名 ​​中表示为变量"_lp"）时，如果道钉是三聚体复合物，3次对称性应该是显而易见的。 </li><li>第三阶段。这个阶段的目的是进一步缩小视图四周矢量的角度与正确的对称性。输入文件是步骤3.2.9输出SEL文件。的参数是相同的，在步骤3.2，只有少数例外: <ol><li>让周围的秒杀观察向量的夹角适当的修改。例如，如果该结构具有3倍的对称性，允许以60度的α角的变化（参数"--alphalimit 60"）。 </li><li>适用于平均值（参数--symmetry C3"）的正确对称（ 如 C3）。 </li><li>使用最后一次迭代生成的两个MAP文件步骤3.2.9（文件名记为标签"，甚至"与"奇"）作为输入模板文件（参数"--Template1"和 --Template2）。 </li><li>运行5-10重复排列和平均（ 例如 ，参数"--firstiter 16 --lastiter 25"）。监视中的变化报道的分辨率。当没有显著变化观察到的，该过程可以停止。 </li></ol></li><li>第四阶段。这个阶段的目的是要精确地同时缩小所有三个角度。输入文件是步骤3.4.4输出SEL文件。的参数是相同的，在步骤3.4，只有少数例外: <ol><li>允许在围绕鞋钉视图向量（参数" - alphalimit 8"）的角8度的改变和在穗视图向量的方向8度的变化（参数"--thetaphilimit 8"）。完善的方向，以4级精度（参数"--angles 8,8,8 --iterate 2"）。 </li><li>允许较小的变化（ 比如 ，5个像素），来解释在先前的校准步骤的误差（参数"--shiftlimit 5"）。 </li><li> ü硒在步骤3.4.4最后一次迭代生成的两个MAP文件（通过标签"，甚至"表示，并在文件名"奇"）作为输入模板文件（参数"--Template1"和--Template2）。 </li><li>运行5-10重复排列和平均（ 例如 ，参数"--firstiter 26 --lastiter 35"）。监视中的变化报道的分辨率。当没有显著变化观察到的，该过程可以停止。 </li></ol></li></ol>四代种子的病毒表面的模板匹配和对齐<ol><li>位于生成均匀地涂在病毒表面的模板匹配的种子，并通过运行jviews.py分配一个初始视图向量的种子。使用步骤1.2.2输入文件生成的STAR文件。产生比尖峰的期望数目约1.5倍以上的种子。 注:这一观点矢量接近最接近每个种子点秒杀的方向。<ol><li>要产生均匀分布的种子在近似球形病毒颗粒（参数"--Even"），给半径（参数"--radius"），角距（ 如 20度）的种子（参数"--angle 20" ）和中央的病毒体的坐标。 例如 ，如果病毒体被集中（步骤1.2）中的箱具有250×250×250个像素的大小之后，中央坐标是125125125像素（选项"--origin 125125125"） 。 </li><li>要生成的丝状颗粒均匀分布的种子，使用参数"--Filament"，并同时指定半径和螺旋对称参数（上升和扭曲）。 注:该螺旋对称的参数在这里只是作为界定均匀分布的种子位置的一种便捷方式，他们并不需要反思灯丝尖峰的实际顺序。 </li></ol></li><li>产生的病毒表面explaine两个独立的平均值d在第2节。 <ol><li>产生SEL文件中定义属于集合"1"和"2"使用jsubtomo_evenodd.py和步骤4.1生成的星病毒体文件。 注:为确保数据集的独立性，以前设置的病毒颗粒成团"1"和"2"必须保持不变。 </li></ol></li><li>完善种子的位置。 <ol><li>按照3.1的步骤说明，除非另有说明。使用步骤4.2.1作为输入文件生成的SEL文件。 </li><li>让种子沿法线仅转移至膜（参数"--zshiftlimit"）。调节移位的容许量取决于病毒颗粒从理想球面几何多少偏离（ 例如 ，参数--zshiftlimit 25"）。 </li><li>使用步骤4.2生成的两个MAP文件作为输入模板文件（参数"--Tem（由标签"偶"，并在文件名"奇"表示）板1"和--Template2）。 </li><li>采用独特的后缀，以避免覆盖原输入之星文件（ 例如 ，参数"--suffix _seeds"）。 </li><li>使用的4像素合并以加速计算（参数"--bin 4"）和一个低通滤波器（ 例如 ，参数"--resolution 50"）。 </li></ol></li><li>使用jviews.py（参数"--cmm"）产生精种子之星档案奇美拉标记文件（CMM）。检查种子在嵌合体打开了CMM文件（以及相关的病毒MAP文件）。确保成品种子正确相对于病毒膜对齐。 注意:不同的颜色可以被用于区分标记物的单独套（参数"--color"）。备选地精制标记可以基于它们的交叉相关系数（ 例如 ，参数"--fomcolor 0.1,0.3"）进行着色。 </li></ol> 5，黄金展位ARD迭代对齐和穗粒结构的平均自动找到附近使用精致的瓜子本地模板匹配的病毒分卷的所有尖峰和对齐和平均位于尖峰。使用从手工采摘尖峰初始模板的一个子集所产生的平均水平。 <ol><li>围绕种子进行局部模板匹配以平均使用的所有程序jsubtomo_iterate_gold.py的尖峰。按照步骤3.5的说明，除非另有说明。 <ol><li>输入文件是在步骤4.3生成的种子提炼的SEL文件。 </li><li>为视图矢量角用一个足够大的限制。 例如 ，如果种子产生每20度（步骤4.1），使用的角度大于该值的一半稍大（参数"--thetaphilimit 12"）。让周围的秒杀观察向量的夹角适当的修改。 </li><li>让种子在所述膜的平面内移动（参数"--xyshiftlimit"）。 例如 ，如果种子到种子的距离是25个像素，使用总变速比一半的（ 例如略多，参数"--shiftlimit 6 --xyshiflimit 10"）。 可能需要额外的翻译转移，如果在步骤3.5生成的地图文件都集中在离中心病毒比种子不同的距离:注意。 </li><li>使用步骤3.5生成的两个MAP文件（通过标签"，甚至"表示，并在文件名"奇"）作为输入模板文件（参数"--Template1"和--Template2）。 </li><li>使用步骤3.5（参数--resolution）生成映射文件的当前的分辨率。 </li><li>运行5-10重复模板匹配，对齐和平均（ 例如 ，参数"--firstiter 1 --lastiter 10"）。监视中的变化报道的分辨率。当没有显著变化观察到的，该过程可以停止。 </li><li>每次迭代后，排除重叠的点击率。 例如 ，如果尖峰的宽度为30像素，排除命中是接近了30个像素，以其他的打击（参数"--mindist 30"）。 </li><li>排除安打与低互相关合作效率。例如，包括最好的75％的尖峰对于每个病毒粒子（参数"--topp 75"）。 注:点击数与低互相关合作效率很可能代表假阳性命中。 </li></ol></li></ol> 6，可视化的结果<ol><li>在嵌合体可视化开启秒杀的精致结构和装修的原子结构。 <ol><li>使用在最后一次迭代产生的低通滤波后的MAP文件（用标记"LP"），在步骤5.1.6作为输入文件。 </li><li>使用基于在嵌合体装修交相关步骤5.1.6所示的决议"适合的地图"的工具。 </li></ol></li><li>创建virio的复合模式N使用jsubtomo_create_model.py。 <ol><li>使用在最后一次迭代产生的低通滤波后的MAP文件（用标记"LP"），在步骤5.1.6作为输入的MAP文件（参数"--template"）。 </li><li>在步骤5.1.6作为输入之星文件使用在最后一次迭代产生之星文件。 </li><li>使用步骤3.2.1生成的模板文件占的复合模式（参数"--mask"）重叠的密度。 </li><li>表明病毒粒子MAP文件的大小，以产生相同的大小的复合模型（参数"--size"）。 </li></ol></li><li>打开复合模型进行可视化的嵌合体 。 </li></ol>

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The authors have nothing to disclose.

<html>知识的病毒糖蛋白的尖峰结构对病毒体膜是用于理解病毒的复制和显影疗法，治疗和预防感染是必不可少的。电子低温显微术结合单个颗粒的平均已成为使用最多的方法来解决包膜病毒粒子，包括糖蛋白的尖峰的结构。然而，该方法仅限于icosahedrally对称的病毒。在这里，通过低温电子断层扫描和subtomogram平均在Jsubtomo的应用程序，我们已经提出了一个通用协议，用于确定糖蛋白尖峰形性包膜病毒是不适合其他流动结构生物学的方法。我们的代表性结果证明，该方法的分辨率足以揭示见解域架构，低聚，并在完整的病毒颗粒的糖蛋白尖峰高阶组织。 jove_content"&gt;这个协议中最关键的一步是兴建两座可靠的启动模式，在统计上是相互独立的。成功执行此步骤假定糖尖峰足够大，不装反目成仇得太紧，使个人尖峰在视觉上识别和手动拾取的断层图像，以及两个独立的模式取平均。如果这是不可行的，两个修饰的协议可以尝试。首先，两个独立的随机模型可通过首先定义subtomograms两个随机子集，然后构造这些子集30内平均该subtomograms。其次，如果孤立穗的结构是来自通过其他手段，例如通过X射线晶体结构，它可以被用作初始模型，然而，必须小心到低使用低分辨率截止（50-70）带通滤波器这种模型中，如产生的两个模型在下一回合的细化将是统计独立只有超过这个分辨率。由于这个警告，前一种方法的建议。 从该协议中获得的分辨率取决于四个主要因素:1。数据收集策略和所述输入数据的质量，ⅱ。该subtomograms的数目，三。该subtomograms的对准精度，以及iv。异质性的结构。而第一和第二限定可以在很大程度上克服通过使用高的信号 - 噪声直接电子检测器结合的CTF校正成像和自动数据采集，对准精度进一步受到关注自身结构的大小和形状。当应用该协议上缺乏显着的特征的小尖峰，它可能是有利的Fab片段结合于尖峰提高对准精度，因而分辨率31。最后，如​​果结构被平均展出多个构象，子tomogr时的分类方法可用于分别平均不同的构象。为此，Jsubtomo集成了发电机包，提供强大subtomogram分类9。 上述协议是互补的分离的病毒糖蛋白的X射线晶体结构。晶体结构可以被嵌入到子断层图像的平均值，得到的糖蛋白的精确取向相对于所述病毒体膜。这种方法的应用，无疑将继续脱落到包膜病毒的结构和病理学的光。

电子冷冻断层扫描是一种电子低温显微成像技术允许一个三维（3D）重建的复杂生物样品的计算。合适的样本范围从纯化的大分子复合物1中 ，单丝2，被小泡3，和多形性膜病毒4至整个原核细胞5，甚至薄整个真核细胞6的区域。以下的倾斜系列，3D断层扫描卷或断层图像的数据收集，可以使用几种建立软件包，包括Bsoft 7和IMOD 8来计算。 固有的生物标本通过电子低温断层限制相应的断层卷的生物学解释的研究两个方面。首先，由于可应用到生物材料引入显著辐射损伤之前的有限的电子剂量，Signa的升噪比在断层数据通常是非常低的。其次，由于数据收集过程中有限样本的倾斜几何的结果，一些意见的对象仍然存在，导致在断层容积所谓的"缺少楔"神器。然而，无论这些限制可以如果断层容积包含重复相同的结构，如大分子复合物，可以成功地平均9-12来克服。 之前，均从断层图像的重建结构中，感兴趣的对象，必须找到并对齐，以相同的方向。定位这种结构可以通过一个模板结构中的断层容积的互相关使用通常被称为模板匹配13的方法来实现。在这个匹配过程中使用的模板可以从电子低温显微术或电子低温层析结合3D重建中得到，或者它可以是从模拟的密度图原子结构。一些计算软件包已经开发完成这些任务11。 平均膜病毒，如HIV-1糖蛋白尖峰，一直是特别成功的方法来研究它们的结构14-16。该结构的理解是积分揭示病毒 - 宿主相互作用的两个分子基础和指导抗病毒和疫苗的设计开发。而大分子晶体是首选的高分辨率个别病毒糖蛋白及其复合物的结构分析（通常大于4埃更好）的技术中，从该方法中产生的X射线结构是蛋白质的天然膜环境中分离的病毒颗粒的。因此，重要的信息，如病毒糖蛋白的高次结构，在病毒粒子的情况下，仍然缺乏。另一方面，电子低温显微术和单粒子重建的整个包膜病毒被限制到病毒粒子具有二十面体对称17,18。电子cryotomography结合分卷排列也因此成为一个互补的技术，允许形状不规则， 在原地多形病毒糖蛋白尖刺的研究。 我们已经开发了一个名为Jsubtomo（www.opic.ox.ac.uk/jsubtomo）的断层分卷的检测，校准和平均软件。 Jsubtomo已被用于一些细胞和病毒结构19-26的结构测定。在这里，我们勾勒出一个详细的协议，这使得测定病毒表面刺突结构。要通过相关的噪声规避过度细化的平均结构中，"黄金标准"细化方案采用10,27。最后，对于可视化的典型结果的解释策略进行了讨论。

<table><tbody><tr><td>Jsubtomo (ver 1.3.1)</td><td>University of Oxford</td><td>n/a</td><td>www.opic.ac.uk/jsubtomo</td></tr><tr><td>Bsoft (ver 1.8.7)</td><td>NIAMS, NIH</td><td>n/a</td><td>bsoft.ws</td></tr><tr><td>UCSF Chimera</td><td>UCSF</td><td>n/a</td><td>www.cgl.ucsf.edu/chimera</td></tr></tbody></table>

averaging of viral envelope glycoprotein spikes from electron cryotomography reconstructions using jsubtomo

包膜病毒利用在其表面上的膜糖蛋白介导进入宿主细胞。这些糖蛋白&#39;尖峰&#39;的三维结构分析通常是在技术上具有挑战性，但重要的是了解病毒的发病机制和药物设计。在这里，一个协议提交病毒刺突结构的决心，通过低温电子断层扫描数据计算平均值。低温电子断层扫描是电子显微镜技术来获得三维断层容积重建，或断层，多形性生物样本，如在接近本机，冷冻水合状态的膜病毒。这些断层图像揭示的利益结构在三维空间中，虽然在低的分辨率。子体积，或子断层图像的计算平均值，是必要的，以获得重复的结构基序，如病毒糖蛋白的尖峰的更高分辨率的细节。阿里的详细计算方法gning和平均使用Jsubtomo软件包子断层概述。这种方法使病毒糖蛋白的尖峰结构的可视化高阶尖峰对尖峰上的病毒膜相互作用的研究在20-40范围内的分辨率和研究。典型的结果示于Bunyamwera病毒，从家庭布尼亚病毒科的包膜病毒。该系列是病原体威胁到人类和动物的健康构成威胁一个结构多元化的群体。

我们证明上面使用此前公布的数据Bunyamwera病毒（Orthobunyavirus， 布尼亚病毒科 ）的包膜糖蛋白复合物中列出的子断层扫描平均工作流程设置24应用程序。数据收集和精化参数列于表1中 ，其中一个代表断层图像示于图2。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 参数（单位） </td><td> 价值 </td></tr><tr><td> 数据收集 </td><td></td></tr><tr><td>电压（kV） </td><td> 300 </td></tr><tr><td>校准放大倍数（倍） </td><td> 111,000 </td></tr><tr><td>像素尺寸（） </td><td> 5.4 </td></tr><tr><td>估计量（E - / 2） </td><td> 100 </td></tr><tr><td> Underfocus（微米） </td><td> 4.0-4.5 </tD&gt; </tr><tr><td>首先周大福零（一） </td><td> 26-30 </td></tr><tr><td>倾斜范围（°） </td><td> -60-60 </td></tr><tr><td>倾斜采样（°） </td><td> 3 </td></tr><tr><td> 数据和细化 </td><td></td></tr><tr><td>断层</td><td> 11 </td></tr><tr><td>病毒体</td><td> 29 </td></tr><tr><td>每个病毒种子</td><td> 106 </td></tr><tr><td>种子总数</td><td> 3074 </td></tr><tr><td>尖峰检测</td><td> 1,346 </td></tr><tr><td>除去重叠后，尖峰</td><td> 1,401 </td></tr><tr><td>互相关为基础的选择后的尖峰</td><td> 1,022 </td></tr><tr><td>包括在最终的平均尖峰</td><td> 1,022 </td></tr><tr><td>对称</td><td> C3 </td></tr><tr><td>角度采样（°） </td><td> 8 </td></tr><tr><td>分辨率在细化使用范围（A） </td><td> 42-334 </td></tr><tr><td>最后的解决估计（A）B </td><td> 35 </td></tr></tbody></table> 表1:Bunyamwera数据收集和细化的统计数据。 à周大福，对比传递函数。 b。使用在0.143阈值两个独立的精致结构之间的傅立叶壳的相关计算。 <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig2highres.jpg" width="600" /> 图2:通过Bunyamwera病毒体的断层图像切片中的每个病毒粒子的外周明显若干尖峰的侧视图来表示带箭头。的断层图像已经经过低通滤波，以60埃。比例尺为100nm。 首先，我们使用精制205人工采摘尖峰初始模型（ <strong&gt;图3）。最中心的尖峰的三次对称性是不施加任何对称性（ 图3B）明显和被强加在随后的几轮细化（ 图3C）。对于病毒粒子表面上自动地检测所有的尖峰，我们产生106种对每个病毒粒子在43 nm和20度（ 图4A）的间距半径和迭代细化它们的位置相对于所述膜（ 图4B）。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig3highres.jpg" width="600" /> 图3:起始模板结构的细化。 （一）圆柱的平均尖峰手动定义的位置建造（C100）模板（B）平均为密度经过五轮的细化，没有任何对称（C）施加显示器秒杀了三倍对称的特点。该模型的分辨率为48。穗（三）平均在41后的五轮细化了三次对称性解决。 <img alt="图4" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig4highres.jpg" width="600" /> 图4:将种子细化AB）的种子之前（A的一个子集）和之后（B）的细化是从图2所示的一个病毒粒子密度在（B）的种子已经颜色编码基于各个交叉。相关系数（蓝色，低相关性;红色，高的相关性）。 最优相关糖蛋白刺突补丁（顶部75％的去除重叠后;〜1,000尖峰）被用于计算最终的平均值。平均得到解决，以35的分辨率（ 图5）。它揭示了三聚尖峰结构的中间，在除了从六个邻近的尖峰一定的贡献。的病毒粒子，通过将结构中的已知位置来计算的复合模型，显示在病毒粒子表面上（ 图6A）中的尖峰的位置。偶尔，尖峰局部有序的补丁是很明显的（ 图6B）。 <img alt="图5" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig5highres.jpg" width="600" /> 图5:模板匹配后的糖蛋白刺突层的贴片精致结构。 （AB）两地图"偶"和"奇"，从数据的两半重建示。这两个图显示一个显着的相似程度，验证了该方法的合法性验证。围绕穗长轴线的方向是不同的两个映射已经重建完全彼此独立。这两个地图（C）平均显示在35埃的最终解决。 <img alt="图6" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig6highres.jpg" width="600" /> 图6:在Bunyamwera病毒尖刺的放置。 （一）病毒体的复合模式。查看向量（支）表示尖峰的方向。颜色表示每个穗和模板结构（蓝色，低相关性;红色，高相关）之间的互相关（B）一个特写尖峰的有序贴剂。

Watch this Scientific Journal Video about Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo at JoVE.com

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

使用Jsubtomo从电子Cryotomography重构平均病毒包膜糖蛋白穗

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

averaging-viral-envelope-glycoprotein-spikes-from-electron

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

12.1K 次观看.  University of Oxford. An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

视频: Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

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

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

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.
In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

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 ...

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.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

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 ...

生物工程

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 ...

生物化学

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 ...

Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It is extremely desirable to pair high-resolution cryo-EM information with dynamic observations as many events occur at rapid timescales - in the millisecond range or faster. Improved knowledge of flexible structures can also assist in the design of novel reagents to combat emerging pathogens, such as SARS-CoV-2. More importantly, viewing biological materials in a fluid environment provides a unique glimpse of their performance in the human body. Presented here are newly developed methods to investigate the nanoscale properties of virus assemblies in liquid and vitreous ice. To accomplish this goal, well-defined samples were used as model systems. Side-by-side comparisons of sample preparation methods and representative structural information are presented. Sub-nanometer features are shown for structures resolved in the range of ~3.5-&#197;-10 &#197;. Other recent results that support this complementary framework include dynamic insights of vaccine candidates and antibody-based therapies imaged in liquid. Overall, these correlative applications advance our ability to visualize molecular dynamics, providing a unique context for their use in human health and disease.

Here protocols are described to prepare virus assemblies suitable for liquid-EM and cryo-EM analysis at the nanoscale using transmission electron microscopy.

推进液体和冰中病毒组件的高分辨率成像

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It ...

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 ...

生物学

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

与VirusMapper超分辨率显微镜开源单粒子分析

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

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 ...

使用Jsubtomo从电子Cryotomography重构平均病毒包膜糖蛋白穗

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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

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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

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摘要

探索更多视频

此视频中的章节

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

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

使用Tomoauto:一个协议，用于高通量自动化低温电子断层扫描

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

推进液体和冰中病毒组件的高分辨率成像

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

ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

与VirusMapper超分辨率显微镜开源单粒子分析

冷冻电子断层扫描远程数据收集和断层扫描平均

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