STRUCTURE OF CLATHRIN CAGES BY STEM AND METAL REPLICATION

正在建立方法来表征结构和 体外组装分子筛三价离子和分子筛笼子的研究 我们对受体介导的内吞作用的能量学的理解。 不同种类笼状蛋白大分子组装体的制备 缓冲液被吸附在以EM为载体的薄碳衬底上 然后在零下180摄氏度的液态乙烷中快速冷冻。 样本要么被冷冻转换成高分辨率的 用于测量分子量分布的场发射STEM， 或冷冻转移到真空室中进行冷冻蚀刻 铂/碳阴影。有必要确定 冷冻干燥和金属阴影达到最佳状态的条件 决议。金属复制品在传输中以数字方式成像 配备慢扫描致冷CCD摄像机的电子显微镜 由Power Macintosh电脑控制。数字电子显微镜 具有提供可立即访问的数据的优势，而无需 需要一间黑暗的房间。

Methods are being established to characterize the structure and assembly of clathrin triskelions and clathrin cages in vitro to help our understanding of the energetics of receptor-mediated endocytosis. Preparations of the clathrin macromolecular assemblies in various buffers were adsorbed onto thin carbon substrates supported on EM grids, then rapidly frozen in liquid ethane at -180 degrees C. Specimens are either cryotransferred into a high-resolution field-emission STEM for measurement of molecular-weight distributions, or cryotransferred into a vacuum chamber for freeze-etching and platinum/carbon shadowing. It has been necessary to determine the conditions for freeze-drying and metal shadowing to achieve optimal resolution. The metal replicas are imaged digitally in a transmission electron microscope equipped with a slow-scan cooled CCD camera controlled by a Power Macintosh computer. Digital electron microscopy has the advantage of providing immediately accessible data without the need for a dark room.