Methods for the Analysis of Helical Macromolecular Complexes

描述（由申请人提供）：螺旋聚合物是由细菌，古细菌和真核细胞中的许多蛋白质形成的，也可以作为病毒衣壳，核衣壳和尾部存在。在某些情况下，细胞中发现的大多数蛋白质以螺旋聚合物的形式存在，因此研究这种聚合物的结构和动力学的方法具有很大的普遍意义。特定的螺旋复合物，如致病菌的毛和鞭毛丝，与人类疾病有非常直接的关系。我们建议进一步发展、扩展、应用和支持从电子显微镜图像中三维重建这些聚合物的新方法。我们在这一领域的工作已经对美国国立卫生研究院支持的许多实验室项目产生了重大影响，但我们现在正处于一个转折点，从大量样本中获得高分辨率结构的潜力现在相当高。鉴于Titan Krios TEM即将到来，我们需要支持进一步开发我们的迭代螺旋实空间重建方法，并优化大量高分辨率聚合物图像的处理。将努力投入使用新的算法进行对齐和重建，以及开发利用从同一灯丝切割的不同部分之间的空间关系的先验知识的方法。所有的开发工作都将使用对广大社区有极大兴趣并与人类健康有直接关系的样本。这些范围从细菌毛到病毒衣壳再到致癌基因的蛋白质产物。我们已经确定，这类图像的处理可以随着处理器数量的增加而扩展，因此我们将努力使这些程序在相对便宜和商业可用的集群上易于使用。将开发用于检测螺旋对称中潜在歧义的工具，但主要工具将是将螺旋重建的分辨率提高到可以识别二级结构的程度。在这个决议下，这些歧义消失了。

DESCRIPTION (provided by applicant): Helical polymers are formed by many proteins found in bacterial, archaeal and eukaryotic cells, and can also be present as viral capsids, nucleocapsids and tails. In certain cases most of the protein found in a cell is in the form of a helical polymer, so methods to study the structure and dynamics of such polymers have great general interest. Particular helical complexes, such as the pili and flagellar filaments of pathogenic bacteria, have a very immediate relevance to human disease. We propose to further develop, extend, apply and support new methods for the three-dimensional reconstruction of such polymers from electron microscopic images. Our work in this area has already had a large impact on a number of NIH-supported projects from many laboratories, but we are now at a transition point where the potential for achieving high- resolution structures from numerous samples is now quite high. Given the imminent arrival of a Titan Krios TEM we need support to further develop our Iterative Helical Real Space Reconstruction approach and optimize the processing of large numbers of high-resolution images of polymers. Effort will be invested in using new algorithms for alignment and reconstruction, as well as in developing methods that make use of prior knowledge about the spatial relations between different segments that have been cut from the same filament. All of the development work will use samples that have great interest to a large community and that have a direct relation to human health. These range from bacterial pili to viral capsids to the protein product of an oncogene. We have established that processing of such images can scale with the number of processors, so effort will be invested in making these programs easy to use on relatively inexpensive and commercially-available clusters. Tools for detecting potential ambiguities in helical symmetry will be developed, but the main tool will be bringing the resolution of helical reconstructions to the point where secondary structure can be recognized. At this resolution these ambiguities disappear. PUBLIC HEALTH RELEVANCE: Many bacteria that cause human diseases require helical polymers on their surface for both attachment and motility. Helical viruses also exist, as do helical polymers formed by proteins from HIV and other pathogenic viruses. Developing improved methods to study the structure of helical polymers will thus have a direct impact on understanding and preventing many diseases.