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。

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.