Name: single particle electron microscopy reconstruction of the exosome complex using the random conical tilt method
Uploaded: 2011-03-28T14:00:00
Description: Watch this Scientific Journal Video about Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method at JoVE.com

作者要感谢在加州大学伯克利分校诺加利斯实验室的成员，在帮助建立初始协议，并在耶鲁大学在他们的帮助王实验室的成员，以建立完整的协议。我们也承认在冷冻电镜设施，并在耶鲁大学医学院的支持高性能计算中心的工作人员。硬件是史密斯一家得奖。

1。随机锥形倾斜方法的原理<ol><li>的随机锥形倾斜方法的原则要求，采取对同一地区内的电子显微镜对标本的显微。一张图片是采取标本untilted位置（图1A）和其他图片是在50至70度（在我们的例子中，我们使用55度）之间的角度倾斜标本。 （图1B） </li><li>使用计算机，数字化显微镜对提出的方方和相同的粒子图像被选中。 （图1C） </li><li> untilted粒子及其倾斜伙伴的图像在三维坐标，彼此相关，?...

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在这篇文章中，我们提出了一个详细的协议的样品制备和3负染色电子显微镜的外切体的使用复杂的三维重建。使用这种方法，我们获得了使用随机锥形倾斜的方法没有任何先验知识结构的三维重建。随机锥形倾斜的方法并不一定要求均匀的样品，但下面的投影匹配的细化步骤需要均匀的试样，以实现高分辨率。 负染样品，污渍的厚度是非常重要的随机的锥形倾斜的方法来工?...

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single particle electron microscopy reconstruction of the exosome complex using the random conical tilt method

单粒子电子显微镜（EM）重建最近已成为一种流行的工具来获得大量的大分子复合物的三维（3D）结构。 X射线晶体相比，它具有一些独特的优势。首先，单粒子电磁重建不结晶的蛋白质样品，这是在X射线晶体学的瓶颈，尤其是对大量的大分子复合物。其次，它并不需要大量的蛋白质样品。单粒子电磁重建毫克蛋白质结晶必要相比，只需要在纳米摩尔浓度的蛋白质溶液的几个微升，使用的EM方法的负染色。然而，尽管少数具有高对称性的大分子集，单粒子EM是有限的分辨率相对较低（低于1纳米的分辨率），尤其是那些没有对称的许多标本。这种技术也由所研究的分子大小的限制，即100 kDa的负染标本和300 kDa的一般冷冻水合标本。 对于一个未知结构的新样品，我们一般使用重金属解决方案嵌入负染色分子。然后，该标本是在透射电子显微镜检查，采取二维（2D）分子的显微照片。理想的情况下，蛋白质分子有一个同质化的三维结构，但表现出不同方向的显微照片。这些显微照片的数字化和计算机处理“单粒子”。用二维的调整和分类技术，在相同的看法同质分子聚集成类。其平均值提高的信号分子的2D形状。我们分配适当的相对取向（欧拉角）的颗粒后，我们将能够2D粒子图像重建成三维虚拟卷。 在单个粒子的三维重建，一个必不可少的步骤是正确分配每个单粒子的正确方向。有几种方法来分配每个粒子的看法， 包括 1角的重整和随机的锥形倾斜（RCT）方法2。在这个协议中，我们描述了在我们的实践，得到酵母外切体采用负染色EM和Rct复杂的三维重建。应该指出的是，我们的电子显微镜和图像处理的协议如下RCT基本原则，但不是唯一的方法来执行的方法。我们首先介绍如何嵌入到一个铀酰甲酸层的厚度相媲美的蛋白质大小的蛋白质样品，使用多孔碳电网覆盖着一层连续的薄碳膜。 untilted（0度）和倾斜（55度），将用于处理和获取初始酵母外切体的三维模型后的显微对，然后插入透射电子显微镜，收集标本。为此，我们进行RCT和再细化匹配细化方法3使用投影初始的3D模型。

Watch this Scientific Journal Video about Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method at JoVE.com

Single Particle Electron Microscopy Reconstruction of the Exosome Complex Using the Random Conical Tilt Method

本文介绍了一种标准的方法来获得一个生物大分子的三维（3D）重建使用负染色电子显微镜（EM）的。在这个协议中，我们将解释如何使用随机的锥形倾斜重建方法（RCT）的决议中等酿酒酵母外切体复杂的三维结构。

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