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标题：Suspending samples over carbon holey films increases heterogeneity of molecular orientations in negative stain electron microscopy
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作者：Katayoun Mansouri ; Robert J. Edwards
期刊名称：MethodsX
印刷版ISSN：2215-0161
电子版ISSN：2215-0161
出版年度：2019
卷号：6
页码：582-586
DOI：10.1016/j.mex.2019.02.032
语种：English
出版社：Elsevier
摘要：Graphical abstractDisplay OmittedAbstractNegative stain electron microscopy (NSEM) is a simple and cost effective method to analyze a wide variety of specimens, especially proteins. In traditional NSEM, the protein sample is applied to and supported by a continuous carbon film. Unfortunately, many proteins stick to the carbon film with a limited number of orientations. Because the restricted orientation limits the available views of the molecule, information about the three-dimensional structure of the molecule is likewise limited. The method presented here overcomes this limitation by using a carbon holey film combined with 1-octadecanol as a spreading agent. We demonstrate this method with solubilized envelope (Env) proteins from HIV, which typically show a restricted orientation on continuous carbon film, and show the following:•1-octadecanol added to negative stain aids the formation of a continuous sample-stain layer spanning the holes of a holey carbon film.•Samples negatively stained over holes show less restricted orientation, resulting in better single particle reconstructions.
关键词：1-Octadecanol;Uranyl formate;Carbon film;Preferred orientation;Single particle reconstructions