DEVELOPMENT OF LOW TEMPERATURE AFM, FREEZE FRACTURE/ETCH

低温 AFM、冷冻断裂/蚀刻的发展

• 批准号: 2283576
• 负责人: ZHIFENG SHAO
• 金额: $ 12.59万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-15 至 1996-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2283576
• 关键词: atomic force microscopy; biomedical equipment development; conformation; cryoscience; freeze etching; membrane proteins; protein structure

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.

阐明蛋白质构象变化及其相互作用， 沿着更高的有序结构，已经成为重要的和令人兴奋的目标 结构生物学研究。 但是，一般适用的 非晶态结构测定技术 高分辨率（亚纳米范围）的大分子还没有被 开发 原子力显微镜不需要薄的或薄的 晶体样品，并具有原子分辨率的潜力，有限 只有通过软材料的变形。 本项目的主要目的 是开发一种在液氮中操作的原子力显微镜 温度 这种低温原子力 具有良好的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， 相关的大分子，在低温下稳定抗变形 温度下，分辨率优于1 nm。 的帮助下 不同的标本制备技术，甚至纯化非唇 可以在这样的系统中研究重构的大分子。 在 原则上，没有结晶和/或样品操作，例如 在该技术中需要固定、拉紧或制作复制品。 三年的项目期将用于设计和建造 液氮温度原子力显微镜， 环境压力。 这是一种新颖的方法， 易于实施（与基于特高压的系统相比）， 超干净的工作环境。 完整的系统，包括 操作的环境压力冷冻断裂/冷冻蚀刻设备， 液氮（压裂）和/或纯氮蒸气（压裂和 蚀刻），将容纳在一个良好的绝缘杜瓦瓶与挡板组件 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 交换标本。 其他成像方法，如共聚焦显微镜 和电子显微镜，将被广泛用作补充 技术，以确保仪器以最小的 藏物 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 可以用其他目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 对我们理解许多重要的生物过程和机制 基于大分子的构象变化。