Obtaining 3D structures with subnanogram macromolecules

描述（由申请人提供）：生物大分子的结构研究总是从生产微克至毫克数量的分子开始，适用于单分子电子显微镜（EM）、X射线晶体学或NMR测量。对于亚纳克量的生物大分子，使用当前技术获得它们的三维（3D）结构是极其困难的。我们建议开发创新技术，克服数量限制，并使用单分子EM从亚纳克数量的生物复合物中获得3D结构。关键的创新将是开发基于亲和力的方法，将低丰度的生物大分子特异性地富集到碳膜上，并为单分子EM和3D重建做好准备。该技术的可行性将通过引入特定的生物配体的表面的碳薄膜和评估的同源生物复合物的选择性结合，并通过应用镍螯合表面的His标记的蛋白质复合物（KvAP/Fv）的富集计算其三维结构进行检查。为了测试对低丰度复合物的应用，我们将功能性人端粒酶复合物富集到修饰的碳膜表面并产生其第一个3D结构。总之，新技术的成功发展将使人们有可能产生许多多组分复合物的结构，这些复合物仅在亚纳克水平上可用。公共卫生相关性：利用目前可用的技术，我们需要在考虑获得其三维（3D）结构之前将大分子纯化到微克至毫克水平。对于亚纳克数量的材料，几乎不可能获得目标分子的3D结构。单粒子电子显微镜从数千个单个分子的图像中计算3D结构。有了数以百万计的分子图像，现在可以实现亚纳米到原子分辨率的结构。这意味着，一旦我们知道如何对数百万个分子进行成像，就可以用亚纳米材料获得结构。这项提议旨在开发新技术，使我们能够从亚纳克生物材料中拍摄数百万分子的图像。它将使用纳米级的化学反应将特定的配体锚在基底表面（惰性碳膜）上，并选择性地结合只能预纯化到约0.1纳克水平的低丰度生物大分子。然后将这些分子准备用于EM成像和其结构的重建。这项新技术将把结构研究扩展到许多重要的大分子复合物，这些复合物难以大量生产，但在各种细胞功能中起着关键作用。

DESCRIPTION (provided by applicant): Structural studies of biological macromolecules always start with producing the molecules in microgram to milligram quantities suitable for single molecule electron microscopy (EM), X-ray crystallography or NMR measurement. With subnanogram amount of biological macromolecules, it is prohibitively difficult to obtain their three-dimensional (3D) structures using the current technology. We propose to develop innovative technology that will overcome the quantity limit and obtain 3D structures from subnanogram quantities of biological complexes using single molecule EM. The key innovation will be to develop affinity-based methods that will specifically enrich low-abundance biological macromolecules onto carbon films and prepare them for single molecule EM and 3D reconstruction. The feasibility of this technology will be examined by introducing specific biological ligands to the surface of thin carbon films and evaluating the selective binding of cognate biological complexes, and by applying a Ni-chelating surface to the enrichment of a His-tagged protein complex (KvAP/Fv) for calculating its 3D structure. To test the application to a low- abundance complex, we will enrich functional human telomerase complex to the surface of modified carbon films and generate its first 3D structure. In summary, successful advancement of the new technology will make it possible to generate structures of many multi-component complexes only available at subnanogram levels. PUBLIC HEALTH RELEVANCE: With the currently available technology, we need to purify macromolecules at microgram to milligram levels before thinking of obtaining their three-dimensional (3D) structures. With subnanogram quantity of materials, it is almost impossible to obtain 3D structures of target molecules. Single particle electron microscopy calculates 3D structures from thousands of images of individual molecules. With millions of molecular images, it now becomes achievable to generate structures at subnanometer to atomic resolutions. This means that it is feasible to obtain structures with subnanogram materials once we know how to image millions of molecules. This proposal is aimed at developing new technology that will enable us to take images of millions of molecules from subnanogram biological material. It will use nano-scale chemical reactions to anchor specific ligands onto a substrate surface (inert carbon film), and bind selectively low-abundance biological macromolecules that can only be pre-purified to ~0.1 nanogram level. These molecules will then be prepared for EM imaging and reconstruction of their structures. The new technology will expand the structural study to many important macromolecular complexes that are difficult to produce in large quantities but play pivotal roles in various cellular functions.