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首页> 外文期刊>Current Protocols in Microbiology >Negative-Stain Transmission ElectronMicroscopy of Molecular Complexes forImage Analysis by 2D Class Averaging
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Negative-Stain Transmission ElectronMicroscopy of Molecular Complexes forImage Analysis by 2D Class Averaging

机译：Negative-Stain传输ElectronMicroscopy通过2 d类分子复合物forImage分析平均

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摘要

Negative-stain transmission electron microscopy (EM) is a technique that hasprovided nanometer resolution images of macromolecules for about 60 years.Developments in cryo-EM image processing have maximized the informationgained from averaging large numbers of particles. These developments cannow be applied back to negative-stain image analysis to ascertain domain levelmolecular structure (10 to 20 A) more quickly and efficiently than possibleby atomic resolution cryo-EM. Using uranyl acetate stained molecular complexesof influenza hemagglutinin bound to Fab 441D6, we describe a simpleand efficient means to collect several hundred micrographs with SerialEM.Using RELION, we illustrate how tens of thousands of complexes can beauto-picked and classified to accurately describe the domain level topologyof this unconventional hemagglutinin head-domain epitope. By comparing tothe cryo-EM density map of the same complex, we show that questions aboutepitope mapping and conformational heterogeneity can readily be answered bythis negative-stain method.
机译：Negative-stain透射电子显微镜(EM)是一个提供纳米技术分辨率图像约60的大分子年。最大化的informationgained吗平均大量的粒子。发展可以应用negative-stain图像分析确定域levelmolecular结构(10 - 20)快速高效地比possibleby原子低温电子显微镜分辨率。分子complexesof流感病毒血凝素绑定到工厂441 d6,我们描述一个简单而有效率意味着收集数百与SerialEM显微图。说明成千上万的复合物beauto-picked和准确的分类描述域层次topologyof这个非传统的血凝素head-domain抗原决定基。通过比较低温电子显微镜密度的地图aboutepitope同样复杂,我们显示问题映射和构象异构性容易回答bythis negative-stain方法。

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