Methods for the Analysis of Helical Macromolecular Complexes

描述（申请人提供）：许多重要的生物大分子以螺旋聚合物的形式存在。例如肌动蛋白、微管蛋白、肌球蛋白、RecA、Rad51、鞭毛蛋白、毛和丝状噬菌体。电子显微镜图像的三维重建的第一个应用是螺旋聚合物，今天一些实验室正在使用完整膜蛋白的螺旋管作为样品，在电子显微镜下解决这些蛋白质的近原子结构。我们开发了一种分析和重建大分子螺旋聚合物电子显微图像的方法。我们可以证明，当存在无序或非均匀性时，当标本衍射弱时，或者当贝塞尔函数重叠时，我们可以用我们的方法比使用传统方法做得更好。在许多情况下，甚至无法进行分析的结构可以使用我们的方法以相当高的分辨率（优于10 A）解决。广泛传播这一方法并进一步发展它，将需要在几个方面作出努力。我们需要创建程序来确定结构是否唯一和正确，并且我们可以显示在有限分辨率下结构确定中存在的模糊性还没有得到充分的认识。我们将需要在一个更加用户友好的EM图像分析工具包中实现该算法，因此使用该方法不需要对SPIDER系统非常熟悉。我们将优化方法的特殊几何形状和问题的脂质管含有完整的膜蛋白。同时，将努力探索一种用于图像对齐和结构细化的实空间算法。所有的方法开发都将使用一些具有重大生物学和临床意义的系统。这些包括丝状噬菌体，在实验室中广泛用作克隆载体和噬菌体展示，来自致病菌的菌毛，被证明对细菌传染性至关重要，来自肠致病性大肠杆菌的III型分泌系统针头，以及f -菌毛，参与细菌结合和细菌群体内抗生素耐药性的传播。因此，这些项目中的大多数将对了解细菌发病机制和疾病的结构产生影响。我们将开发和传播的方法将对美国国立卫生研究院资助的研究的许多领域产生更大的影响，从了解肌肉结构和营养不良到传染病。

DESCRIPTION (provided by applicant): Many important biological macromolecules exist as helical polymers. Examples are actin, tubulin, myosin, RecA, Rad51, flagellin, pili, and filamentous bacteriophage. The first application of three-dimensional reconstruction from electron microscopic images was to a helical polymer, and several laboratories today are using helical tubes of integral membrane proteins as specimens for solving the near-atomic structure of these proteins in the electron microscope. We have developed a method to analyze and reconstruct electron microscopic images of macromolecular helical polymers. We can show that when there is disorder or heterogeneity, when the specimens diffract weakly, or when Bessel functions overlap, we can do far better with our method than can be done using traditional approaches. In many cases, structures that were not even amenable to analysis can be solved at fairly high resolution (better than 10 A) using our method. Disseminating this method widely, and further developing it, will require effort along several lines. We need to create procedures to establish whether structures are unique and correct, and we can show that ambiguities that are present in structure determination at limited resolution have not been sufficiently appreciated. We will need to implement the algorithm within a more user-friendly package of EM image analysis tools, so that use of the method does not require extensive familiarity with the SPIDER system. We will optimize the method for the particular geometries and problems of lipid tubes containing integral membrane proteins. At the same time, effort will be put into exploring a real-space algorithm for the alignment of images and refinement of a structure. All of the methods development will take place using a number of systems that are of great biological and clinical interest. These include filamentous bacteriophage, widely used in laboratories as cloning vectors and for phage display, pili from pathogenic bacteria, shown to be essential for bacterial infectivity, Type III secretion system needles from Enteropathogenic E. coli, and F-pili, involved in bacterial conjugation and the spread of antibiotic resistance within a bacterial population. Most of these projects will thus have an impact on understanding structures involved in bacterial pathogenesis and disease. The methods that we will be developing and disseminating will have an even larger impact in many areas of NIH-funded research, from understanding muscle structure and dystrophies to infectious diseases.