DEVELOPMENT OF LOW TEMPERATURE AFM, FREEZE FRACTURE/ETCH

蛋白质构象变化及其相互作用的阐明， 随着更高阶结构的出现，一直是一个重要而令人兴奋的目标 结构生物学研究。然而，一个普遍适用的 非晶态结构的测定技术 高分辨率(亚纳米范围)的大分子还没有 发展起来的。原子力显微镜不需要薄的或 晶体样品，并具有原子分辨率的潜力，有限 只能通过软材料的变形来实现。这个项目的主要目的是 是开发一种在液氮环境下运行的原子力显微镜 温度。这种温度较低的原子力的组合 具有良好冷冻断裂和冷冻蚀刻的显微镜 技术将允许研究膜的精细结构-- 缔合的大分子，在低温度下稳定不变形 温度，分辨率优于1纳米。在…的帮助下 不同的标本制备技术，甚至提纯的无唇 重组的大分子可以在这样的系统中进行研究。在……里面 原理，无结晶和/或样品处理，如 在这项技术中，需要固定、拉伸或复制。 将利用三年的项目期进行设计和施工 液氮温度原子力显微镜 环境压力。这是一种新的方法，因为它相对 易于实施(与基于特高压的系统相比)，并提供 超净的工作环境。完整的系统，包括 常压冷冻压裂/冷冻蚀刻设备在 液氮(破裂)和/或纯氮蒸气(破裂和 蚀刻)，将被安置在带有挡板组件的绝缘良好的杜瓦瓶中 通过扩散消除水污染。低温 部件可远程操作，以减少样本污染并加快速度 向上交换标本。其他成像方法，如共聚焦显微镜 和电子显微镜，将被广泛使用作为补充 技术，以确保仪器以最小的速度运行 手工艺品。 该项目的成功实施将提供一个独特的 研究大分子结构的能力，这不能 使用其他当前可用的技术来实现，例如 电子显微镜和X射线衍射技术，并将有助于 对我们了解许多重要的生物过程和机制 基于大分子的构象变化。

Elucidation of protein conformational changes and their interactions, along with higher order structures, has been important and exciting goal of structural biology research. However, a generally applicable technique for determining the structures of non-crystalline macromolecules at high resolution (sub-nanometer range) has not been developed. Atomic force microscopy does not require a thin or a crystalline specimen, and has the potential of atomic resolution, limited only by deformation of soft materials. The main purpose of this project is to develop an atomic force microscope for operation at liquid nitrogen temperature. Combination of such a lower temperature atomic force microscope with well established freeze fracture and freeze etching techniques will permit the study of fine structures of membrane- associated macromolecules, stabilized against deformation at low temperatures, at resolutions better than 1 nm. With the help of different specimen preparatory techniques, even purified non-lip reconstituted macromolecules can be studied in such a system. In principle, no crystallization and/or specimen manipulation, such as fixation, straining or making replica, are required in this technique. The three year project period will be utilized to design and construct a liquid nitrogen temperature atomic force microscope operating at ambient pressure. This is a novel approach, preferred for its relatively easy implementation (compared with UHV based systems) and providing a super clean working environment. The complete system, including the ambient pressure freeze fracture/freeze etching apparatus operated in liquid nitrogen (fracture) and/or in pure nitrogen vapor (fracture and etching), will be housed in a well insulated dewar with a baffle assembly to eliminate water contamination through diffusion. The low temperature part is remotely operable to reduce specimen contamination and to speed up specimen exchange. Other imaging methods, such as confocal microscopy and electron microscopy, will be used extensively as supplementary techniques, in order to assure that the instrument performs with minimal artifacts. Successful implementation of this project will provide a unique capability for the study of the structure of macromolecules, which cannot be accomplished with other currently available techniques, such as electron microscopy and X-ray diffraction techniques, and will contribute to our understanding of many important biological processes and mechanism based on conformational changes in macromolecules.