Solving macromolecular complex architecture in situ by super-resolution microscop

描述(由申请人提供) 摘要：X射线结晶学、核磁共振和低温电子显微镜等结构生物学方法用于体外测定生物分子的三维结构，对于我们对生物过程的分子理解的快速扩展至关重要。然而，随着挑战转向高阶大分子结构，这些方法都面临着困难，特别是在天然细胞环境中的研究。为了应对这一挑战，我们建议开发一种基于光学显微镜的新方法。在我们最近发展的超分辨光学显微镜技术的支持下，这种方法的关键是通过单分子定位来荧光标记络合物的组成并确定它们的相对位置。为了发展这一方法，我们将通过优化仪器设计、蛋白质标记方法和数据分析算法，进一步提高超分辨率显微镜在分子水平的分辨率。我们还将应用这种方法来研究酵母核孔复合体的结构。下一步，通过对蛋白质单元的各个结构域的位置映射和计算将原子模型匹配到分子络合物的上下文中，我们将获得大分子络合物的全伪原子分辨结构。拟议的研究将建立一个新的结构确定工具，补充现有方法。由于光学显微镜的非侵入性，它唯一的优势是能够在不提纯的情况下原位研究大分子组装。因此，它将极大地帮助理解广泛的大分子组件的结构和结构-功能关系，为了解它们在自然环境中的功能提供洞察，特别是对于那些难以从细胞中提取的组件。 公共卫生相关性：了解生物分子或大分子复合体的结构，就像了解建筑的蓝图一样，为深入了解其作用机制和后续的药物开发奠定基础。这项研究旨在建立一种基于超分辨光学显微镜的新的结构生物学方法，以确定自然环境中大分子络合物的分子结构。它将极大地拓宽可被结构表征的细胞成分的光谱，从而增强我们从机制上理解生物医学问题的能力。

DESCRIPTION (Provided by the applicant) Abstract: Structural biology methods including X-ray crystallography, NMR and cryo-electron microscopy (EM) for determining the three-dimensional structures of biomolecules in vitro were critical to the rapid expansion in our molecular understandings of biological processes. However, as the challenge shifts towards the higher order architecture of macromolecular machineries, these methods all face difficulties, especially for studies in the native cellular environment. To address this challenge, we propose to develop a new approach based on light microscopy. Enabled by our recently developed super-resolution optical microscopy techniques, the key of this approach is to fluorescently label the components of the complex and determine their relative positions through single- molecule localization. To develop this method, we will further improve the resolution of super-resolution microscopy to the molecular level by optimizing the instrument design, protein labeling methods and data analysis algorithm. We will also apply this method to investigating the architecture of yeast nuclear pore complex. As a next step, through position mapping of individual domains of protein units and computation to fit atomic models into the context of molecular complexes, we will obtain full pseudo-atomic resolution structures of large complexes. The proposed research will establish a new structural determination tool that complements existing methods. It is uniquely advantageous is the ability to study large macromolecular assembly in situ without purification, owing to the noninvasive nature of optical microscopy. Thus, it will greatly help understanding the structure and structure-function relationship of a broad range of macromolecular assemblies, providing insights of how they functions in the native environment, especially for those difficult to be extracted from the cell. Public Health Relevance: Understanding the structure of a biomolecule or a macromolecular complex, just like knowing the blueprint of a building, lays the foundation of insights to its function mechanism and subsequent drug development. The proposed research is aimed at establishing a new structural biology method based on super-resolution optical microscopy to determine the molecular architecture of macromolecular complexes in the native environment. It will greatly widen the spectrum of cellular components that can be structurally characterized, thus strengthening our ability for the mechanistic understanding of biomedical problems.