VISUALIZING THE MACROMOLECULAR ORGANIZATION OF THYLACOID MEMBRANES USING CRYO-ET

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。叶绿体的光合膜，被称为类囊体膜，在某种程度上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，人们可以监测体内特定蛋白质复合体的一般功能活性和状态，类囊体内所有主要蛋白质复合体的晶体结构的可用性，以及数百个具有改变的类囊体膜蛋白、类脂、发育模式和结构的突变体的可用性。到目前为止，类囊体膜内蛋白质复合体的最佳分辨率是通过冷冻断裂实现的，这揭示了膜的整体结构。该项目的目的是使用冷冻水化标本，结合电子显微镜断层扫描(ET)，更详细地研究类囊体膜内光合作用机制的大分子组织。使用这种方法，我们不仅有机会将大分子的组织结构与冷冻断裂研究进行比较，而且还可以将分辨率提高到更高的水平，并研究大分子之间是如何相互作用的。这一目标既可以通过观察类囊体膜内和类囊体膜之间的邻居来实现，也可以通过观察这些复合体或其成分的晶体结构如何与低温作用形成的类囊体膜的结构图相匹配来实现。我们已经获得了几种不同的单轴倾斜系列制备物，包括完整的分离的菠菜叶绿体和冷冻切片的菠菜叶绿体，分离的和深度冷冻的类囊体膜，以及完整的衣藻细胞切片。初步结果使我们能够直观地看到类囊体层和基质类囊体面上的ATP合成酶颗粒的组织。我们还发现了尚未鉴定的大分子络合物的准晶排列，类似于冷冻断口图像中看到的有序络合物。

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.