Obtaining 3D structures with subnanogram macromolecules

描述(申请人提供)：生物大分子的结构研究总是从生产适合于单分子电子显微镜(EM)、X射线结晶学或核磁共振测量的微克到毫克量的分子开始。由于生物大分子的量在亚纳克级，利用目前的技术很难获得它们的三维结构。我们建议开发创新技术，克服数量限制，使用单分子EM从亚纳克量的生物络合物中获得3D结构。关键的创新将是开发基于亲和力的方法，这种方法将专门将低丰度生物大分子浓缩到碳膜上，并为单分子EM和3D重建做准备。这项技术的可行性将通过在碳薄膜表面引入特定的生物配体，评估同源生物络合物的选择性结合，以及将镍螯合表面应用于His标记的蛋白质络合物(KvAP/Fv)的浓缩来计算其三维结构来检验其可行性。为了测试低丰度复合体的应用，我们将功能人端粒酶复合体浓缩到修饰的碳膜表面，并生成它的第一个三维结构。总而言之，新技术的成功进步将使人们有可能产生许多仅在亚纳克级水平上可用的多组分络合物的结构。与公共健康相关：利用目前可用的技术，在考虑获得它们的三维(3D)结构之前，我们需要将大分子从微克提纯到毫克水平。在亚纳克级材料的情况下，几乎不可能获得目标分子的三维结构。单粒子电子显微镜从数千张单个分子的图像中计算出3D结构。有了数以百万计的分子图像，现在可以生成亚纳米到原子分辨率的结构。这意味着，一旦我们知道如何成像数百万个分子，就可以获得亚纳克材料的结构。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。它将利用纳米级的化学反应将特定的配体固定在底物表面(惰性碳膜)上，并选择性地结合只能预纯化到~0.1纳克水平的低丰度生物大分子。然后，这些分子将被准备用于电磁成像和重建其结构。这项新技术将把结构研究扩展到许多重要的大分子络合物，这些络合物很难大量生产，但在各种细胞功能中发挥着关键作用。

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.