Methods for Analysis of Helical Macromolecular Complexes

描述(由申请人提供)： 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、药丸和噬菌体尾巴。电子显微镜图像三维重建的第一个应用是螺旋聚合物，现在有几个实验室正在使用完整的膜蛋白螺旋管作为样本，在电子显微镜下解决这些蛋白质的近原子结构。现有的螺旋图像分析和三维重建方法要求螺旋的结构非常规则。不幸的是，许多螺旋聚合物是相当无序的。这可能意味着只有最有序的聚合物才会用现有的方法进行研究。但是，存在于其他标本中的无序，那些无法研究的无序，可能在这些组装的功能中有很大的生物学作用，所以许多有趣的系统可能不适合需要近晶体秩序的方法。此外，当螺旋平均法被施加到可能与这种规则对称性有显著偏差的样品上时，也许更规则的聚合物的结构和动力学中最有趣的方面可能会被掩盖。在过去的一年里，我的实验室开发了一种迭代方法，将单粒子图像处理技术应用于无序螺旋聚合物，并非常成功地应用于参与同源基因重组的肌动蛋白和核蛋白细丝。需要进行密集的开发工作，以使这一方法在其他实验室得到广泛应用。此外，有初步迹象表明，该方法可以如何扩展，使其在分离一组聚合物中的不同结构状态方面更加强大。该项目致力于改进这一方法，并产生一套可供其他调查人员轻松使用的工具。预计这些工具的问世将在细胞生物学、分子生物学和结构生物学等领域产生很大影响。

DESCRIPTION (provided by applicant): Many important biological macromolecules exist as helical polymers. Examples are actin, tubulin, myosin, RecA, Rad51, flagellin, pill, and bacteriophage tails. 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. The existing methods for image analysis and three-dimensional reconstruction of helices require that the structure be very regular. Unfortunately, many of these helical polymers are quite disordered. This may mean that only the most ordered polymers will be studied with existing methods. But the disorder that is present in the other specimens, those that cannot be studied, may have a large biological role in the function of these assemblies, so many interesting systems may not lend themselves to methodologies that require near-crystalline order. Further, perhaps the most interesting aspects of the structure and dynamics of more regular polymers may be obscured when helical averaging is imposed upon a specimen that may have significant deviations from such a regular symmetry. Over the past year an iterative method has been developed in my lab for the application of single-particle image processing techniques to disordered helical polymers, and applied very successfully to actin and nucleoprotein filaments involved in homologous genetic recombination. Intensive development work needs to be done to allow this methodology to be widely used in other laboratories. Further, there are preliminary indications of how the methodology can be extended to make it more powerful in separating out different structural states within a population of polymers. This project is devoted to improving this methodology, and producing a set of tools that can be easily used by other investigators. It is expected that the availability of such tools will have a large impact in fields such as cell biology, molecular biology and structural biology.