CORRELATED IMAGING OF SUPRAMOLECULAR COMPLEXES ANDCELLULAR COMPARTMENTS

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 对结构生物学家的持续挑战是大分子复合物和细胞器的鉴定和结构分析，包括蛋白质、膜和/或核酸的多组分组合，特别是在组织和细胞的背景下。 我们在NCMIR创造并推广了术语“中尺度”（来自希腊语“mesos”，中）作为我们挖掘的成像范围，以获得有关组成细胞，组织和器官内功能复合物的大分子聚集体的结构和动力学的新知识。 这些中尺度结构用光学显微镜成像，并使用电子显微镜以中等分辨率进行更详细的成像，这可以用许多制备方法实现，而不是高分辨率，原子级，3-D结构方法，如核磁共振（NMR），X射线晶体学和分子显微镜（例如电子低温显微镜）。 此外，沿着创建蛋白质-蛋白质相互作用（“相互作用组”）的系统生物学计算方法的任务，人们理想地希望将大分子复合物放置在它们的细胞环境中，以便完全理解它们的功能相互作用（“可视化蛋白质组学”）。 在NCMIR，我们专注于细胞和细胞器的可视化，以及相关光学和电子显微镜（分别为LM和EM）的分子识别。 在这一核心部分，我们使用新的特异性染色策略（四半胱氨酸结构域/双砷配体;量子点标记;特定染色某些蛋白质，碳水化合物或脂质的化学品），我们已经开发了各种生物标本。我们改进了不稳定组织和光转换材料的超微结构保存方法。最后，我们应用先进的数据收集和三维重建方法来推动我们的成像技术的分辨率，以弥合细胞动力学的光显微镜成像和大分子结构的直接可视化之间的差距。作为我们以前的工作的扩展的一部分，我们正在探索新的标本几何形状，将允许360度旋转，因此，随后的断层图像将没有丢失的数据锥，因为目前是定制的标准单轴倾斜或双轴倾斜数据收集板状标本。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A continuing challenge to structural biologists is the identification and structural analyses of large macromolecular complexes and organelles, including multi-component combinations of proteins, membranes and/or nucleic acids, especially within the context of tissues and cells. We at NCMIR have coined and have popularized the term "mesoscale" (from the Greek "mesos", middle) as the imaging range we mine to obtain new knowledge about the structure and dynamics of the macromolecular aggregates that compose the functional complexes within cells, tissues and organs. These mesoscale structures are imaged with light microscopy and with more detail using electron microscopy at moderate resolutions, achievable with numerous preparatory methods, as opposed to high resolution, atomic-level, 3-D structural methods, such as nuclear magnetic resonance (NMR), X-ray crystallography and molecular microscopy (e.g. electron cryomicroscopy). In addition, along with the task of creating systems biology computational approaches to protein-protein interactions (the "interactomes"), one would ideally like to place macromolecular complexes within their cellular environments in order to fully understand their functional interactions ("visual proteomics"). At NCMIR, we focus on the visualization of cells and organelles, and the identification of molecules for correlative light and electron microscopy (LM and EM, respectively). In this core section, we use the new specific staining strategies (tetracysteine domains/biarsenical ligands; quantum dot labeling; chemicals that specifically stain certain protein, carbohydrate or lipids) we have developed for a variety of biological specimens. We improve methods for ultrastructural preservation of labile tissues and photoconverted material. Finally, we apply advanced data collection and 3D reconstruction methods to push the resolution of our imaging techniques in order to bridge the gap between light microscopic imaging of cellular dynamics and direct visualization of macromolecular structure. As part of the extension of our previous work, we are exploring new specimen geometries that will allow for 360 degree rotation and therefore, subsequent tomograms will have no missing cone of data, as presently is the custom for standard single axis tilt or double axis tilt data collection on slab-like specimens.