STRUCTURAL ANALYSIS OF MACROMOLECULAR ASSEMBLIES

图像处理与三维(3-D)新方法 重建使获得结构模型成为可能 复杂的分子组合，如核糖体和 电子分辨率为2-3 nm的血蓝蛋白分子 非晶态样品的显微照片。这一能力将是 用于功能重要配体结合的三维定位 核糖体上的位点和特定蛋白质的定位 核糖体和血蓝蛋白。许多研究将在 与其他实验室的合作。我们还将获得准确的 亚单位：亚单位的模型，分辨率为2-3 nm 70年代(原核生物)和80年代(真核生物)的联合 核糖体通过重组亚基的三维匹配和 核糖体。 具体地说，我们将重建：(1)来自大肠杆菌的50s亚基 L7/L12蛋白耗尽，这些蛋白参与了 与伸长因子相关的GTPase活性 翻译，(2)50S-螺旋霉素-抗体复合体，以 绘制肽基转移酶中心位置图，(3)70年代 含和不含结合tRNA的大肠杆菌单体，(4)a 单特异性抗体标记真核细胞40S亚基 针对其上的一个调节磷酸化位点 蛋白S6，(6)由40S亚基和 启动因子eIF-3，以及(7)哺乳动物的80S核糖体。我们 还将重建24亚基的血蓝蛋白分子 澳洲仙人掌吸虫和一株或多株选定的单抗 免疫复合体来定位特定的亚单位。 这些研究将得到方法学调查的支持。 并通过使用冷冻电子显微镜对冷冻的- 水合的，未染色的标本。

New methods of image processing and three-dimensional (3-D) reconstruction have made it possible to obtain structural models of complex molecular assemblies such as ribosomes and hemocyanin molecules at 2-3 nm resolution from electron micrographs of non-crystalline specimens. This capability will be used in 3-D localizations of functionally important ligand-binding sites on the ribosome and mappings of specific proteins in ribosomes and hemocyanin. Many of the studies will be done in collaborations with other laboratories. We will also obtain precise models, to a resolution of 2-3 nm, of the subunit:subunit association in the 70S (prokaryotic) and 80S (eukaryotic) ribosomes by 3-D matching of reconstructed subunits and ribosomes. Specifically we will reconstruct: (1) the 50S subunit from E. coli depleted of the L7/L12 proteins which are involved in the elongation factor-dependent GTPase activities associated with translation, (2) a 50S-spuromycin-antibody complex in order to map the position of the peptidyl transferase center, (3) the 70S monosome from E. coli with and without bound tRNA, (4) a eukaryotic 40S subunit labeled with a monospecific antibody directed against one of the regulatory phosphorylation sites on protein S6, (6) a complex consisting of the 40S subunit and initiation factor eIF-3, and (7) a mammalian 80S ribosome. We will also reconstruct the 24-subunit hemocyanin molecule from Androctonus australis and one or more selected monoclonal immunocomplexes to localize specific subunits. These studies will be supported by methodological investigations and extended by the use of cryo-electron microscopy of frozen- hydrated, unstained specimens.