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

Авторы хотели бы поблагодарить членов Ногалес лаборатории в Калифорнийском университете в Беркли, помогая установить начальные протоколы и членам Ван лаборатории в Йельском университете в их помощью установить полный протоколов. Мы также признаем, штабов в крио-ЭМ объекта и высокопроизводительный вычислительный центр в Йельской школы медицины за их поддержку. HW является Смит Семья Лауреат премии.

1. Принцип метода случайных Коническая Tilt <ol><li> Принцип случайной конической метод наклона требует принятия пару микрофотографии же области образца внутри электронного микроскопа. Одна картина берется из образца в untilted положение (рис. 1А) и другие съемки образца под углом от 50 до 70 град...

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В этой статье мы представляем подробный протокол пробоподготовки и трехмерные реконструкции exosome комплекса с использованием отрицательных электронной микроскопии окрашивания. Используя этот метод, мы получили 3D-реконструкции с использованием метода случайной конической наклона бе...

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

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large macromolecular complexes. Compared to X-ray crystallography, it has some unique advantages. First, single particle EM reconstruction does not need to crystallize the protein sample, which is the bottleneck in X-ray crystallography, especially for large macromolecular complexes. Secondly, it does not need large amounts of protein samples. Compared with milligrams of proteins necessary for crystallization, single particle EM reconstruction only needs several micro-liters of protein solution at nano-molar concentrations, using the negative staining EM method. However, despite a few macromolecular assemblies with high symmetry, single particle EM is limited at relatively low resolution (lower than 1 nm resolution) for many specimens especially those without symmetry. This technique is also limited by the size of the molecules under study, i.e. 100 kDa for negatively stained specimens and 300 kDa for frozen-hydrated specimens in general.
For a new sample of unknown structure, we generally use a heavy metal solution to embed the molecules by negative staining. The specimen is then examined in a transmission electron microscope to take two-dimensional (2D) micrographs of the molecules. Ideally, the protein molecules have a homogeneous 3D structure but exhibit different orientations in the micrographs. These micrographs are digitized and processed in computers as "single particles". Using two-dimensional alignment and classification techniques, homogenous molecules in the same views are clustered into classes. Their averages enhance the signal of the molecule's 2D shapes. After we assign the particles with the proper relative orientation (Euler angles), we will be able to reconstruct the 2D particle images into a 3D virtual volume.
In single particle 3D reconstruction, an essential step is to correctly assign the proper orientation of each single particle. There are several methods to assign the view for each particle, including the angular reconstitution1 and random conical tilt (RCT) method2. In this protocol, we describe our practice in getting the 3D reconstruction of yeast exosome complex using negative staining EM and RCT. It should be noted that our protocol of electron microscopy and image processing follows the basic principle of RCT but is not the only way to perform the method. We first describe how to embed the protein sample into a layer of Uranyl-Formate with a thickness comparable to the protein size, using a holey carbon grid covered with a layer of continuous thin carbon film. Then the specimen is inserted into a transmission electron microscope to collect untilted (0-degree) and tilted (55-degree) pairs of micrographs that will be used later for processing and obtaining an initial 3D model of the yeast exosome. To this end, we perform RCT and then refine the initial 3D model by using the projection matching refinement method3.

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

В этой статье описывается стандартный метод для получения трехмерных (3D) реконструкция биологических макромолекул с использованием отрицательных электронной микроскопии окрашивания (EM). В этом протоколе, мы объясняем, как получить 3D-структуры exosome комплекс CEREVISIAE Saccharomyces на среднего разрешения с использованием случайного конической метод реконструкции наклона (РКИ).

Одной частицы электронной микроскопии Реконструкция комплекса Exosome Использование случайных Коническая Метод Tilt

Single particle electron microscopy (EM) reconstruction has recently become a popular tool to get the three-dimensional (3D) structure of large ...

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