VISUALIZING THE MACROMOLECULAR ORGANIZATION OF THYLACOID MEMBRANES USING CRYO-ET

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。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。所列机构为中心，不一定是研究者所在机构。叶绿体的光合作用膜，也称为类囊体，在某些方面是研究生物膜许多基本性质的理想模型系统。这些方法包括它们易于分离和纯化，可以监测体内特定蛋白质复合物的一般功能活性和状态，类囊体内所有主要蛋白质复合物的晶体结构的可用性，以及数百种具有改变的类囊体膜蛋白、脂质、发育模式和结构的突变体的可用性。迄今为止，类囊体膜内蛋白质复合物的最佳分辨率是通过冷冻断裂实现的，这揭示了膜的整体组织结构。本项目的目的是使用冷冻水化的标本，结合电子显微镜断层扫描（ET），以更详细地研究类囊体膜内光合机制的大分子组织。通过这种方法，我们不仅有机会将大分子组织与冷冻断裂研究进行比较，而且还将分辨率推向更高水平，并研究大分子之间如何相互作用。这一目标可以通过观察类囊体膜内部和之间的邻居来实现，并通过观察这些复合物或其组分的晶体结构如何适合用cryo-ET开发的类囊体膜的结构图来实现。我们已经获得了几个单轴倾斜系列不同的准备，包括完整的分离，然后冷冻切片菠菜叶绿体，分离和暴跌冷冻类囊体膜，和部分完整的衣原体细胞。初步的结果使我们能够可视化的组织的类囊体堆栈和ATP合酶颗粒的表面上的基质类囊体。我们还发现了尚未确定的大分子复合物的次晶排列，类似于冷冻断裂图像中看到的有序复合物。

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. The photosynthetic membranes of chloroplasts, known as the thylakoids, are in some ways an ideal model system for studying many fundamental properties of biological membranes. These ways include their ease of isolation and purification, the fact that one can monitor general functional activities and the state of specific protein complexes in vivo, the availability of crystal structures of all major protein complexes within the thylakoids, and the availability of hundreds of mutants with altered thylakoid membrane proteins, lipids, developmental patterns and structure. The best resolution of protein complexes within the thylakoid membrane has thus far been achieved via freeze fracture, which has revealed the overall organization of the membrane. The objective of this project is to use frozen-hydrated specimens, combined with electron microscope tomography (ET), to investigate the macromolecular organization of the photosynthetic machinery within the thylakoid membranes in greater detail. With this methodology we not only have a chance to compare the macromolecular organization to the freeze fracture studies, but also to push the resolution to higher levels and investigate how the macromolecules interact with each other. This goal can be pursued both by looking at neighbors within and between thylakoid membranes, and by seeing how the crystal structures of these complexes or their components fit into the structural map of the thylakoid membrane developed with cryo-ET. We have obtained several single-axis tilt series of different preparations, including intact isolated and then cryo-sectioned spinach chloroplasts, isolated and plunge-frozen thylakoid membranes, and sections of intact Chlamydomonas cells. Preliminary results have enabled us to visualize the organization of the thylakoid stacks and the ATP synthase particles on the surface of the stroma thylakoids. We also found paracrystalline arrangements of not-yet-identified macromolecular complexes that resemble ordered complexes seen in freeze fracture images.