Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography.
Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography.
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DOI：
10.3791/53608
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发表时间：
2016-01-30
期刊：
影响因子：
--
通讯作者：
Liu J
中科院分区：
文献类型：
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作者：
Morado DR;Hu B;Liu J
我们提出了一个协议，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原位结构。该协议允许处理大量数据，避免常见瓶颈并减少资源停机时间，使用户能够专注于重要的生物学问题。冷冻电子断层扫描（Cryo-ET）是一种强大的三维（3-D）成像技术，用于在分子水平上可视化大分子复合物。该技术首先通过在玻璃冰中快速冷冻标本来保持样品的天然状态，然后从不同角度收集一系列高倍放大的显微照片，最后通过计算重建三维密度图。冷冻水化样品对电子束极其敏感，因此在非常低的电子剂量下收集显微照片以限制辐射损伤。结果，原始冷冻断层图像具有由固有噪声图像表征的非常低的信噪比。为了更好地可视化感兴趣的对象，利用常规成像分析和子断层图像平均来提高对比度和分辨率，其中从初始断层图像中提取对象的子断层图像并对其进行对准和平均。倾斜系列的大数据集对于理解和解决不同状态、条件或突变的复合体以及获得足够大的子断层图像集合以进行平均和分类是必不可少的。收集和处理这些数据可能是阻止进一步分析的主要障碍。在这里，我们描述了一个高通量的冷冻ET协议的基础上，计算机控制的300千伏冷冻电子显微镜，直接检测设备（DDD）相机和一个高效的，半自动化的图像处理管道软件包装库tomoauto开发的内部。该方案已有效地用于可视化福氏志贺氏菌小细胞中完整的III型分泌系统（T3 SS）。它可以适用于任何适合低温ET的项目。
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. 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.