Low Temperature AFM/Freeze Fracture & Etch

低温 AFM/冷冻断裂

• 批准号: 6325407
• 负责人: ZHIFENG SHAO
• 金额: $ 25.9万
• 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 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6325407
• 关键词: atomic force microscopy; bioimaging /biomedical imaging; biomedical equipment development; conformation; cryoscience; freeze etching; imaging /visualization /scanning; immunoglobulin M; membrane proteins; membrane structure; nanotechnology; nuclear membrane; protein localization; protein structure; structural biology; technology /technique development; virus envelope

DESCRIPTION (provided by applicant): Although a relatively new technique, atomic force microscopy (AFM) has been proven a powerful structural tool in biology. However, despite the successes in acquiring high resolution surface structures of many macromolecules under ambient conditions, the ability of AFM for the study of large, individual complexes that are often exceedingly flexible is still very limited, primarily because of the deformation and damage caused by the scanning tip which exerts a fairly high pressure within the contact area between the tip and the sample. As a result, even with very sharp tips, the resolution has been relatively low on large structures. To overcome this problem and to expand the ability of AFM for the elucidation of molecular structures at the single molecule level, we have developed the first AFM that is operated at the liquid nitrogen temperature under ambient pressure (cryo-AFM). Our results clearly demonstrate that at cryogenic temperatures, the molecular rigidity is significantly improved, allowing clear and high-resolution images to be obtained reliably and reproducibly. With this novel technique, we have been able to reveal structural conformations for several macromolecules that were not known previously. In this continuation application, we seek to continue the development of this new technology for applications in structural biology, with a particular emphasis on single, flexible complexes. We will construct a multi-functional integrated system that will include an array of specimen preparation capabilities. We will also push the technology to improve its resolving power on well preserved, quickly frozen specimens (after deep etching). To achieve this goal, in addition to instrumental improvements, we also plan to utilize the newly developed nanotube tips and "single atom" probes in our cryo-AFM. As an integral part of this project, we will also apply this method to several carefully selected problems, including the structure of IgM, protein distribution in a cell membrane, the surface structure of the nuclear pore complex and an enveloped virus. These applications will provide the initial validation of the methodology, but at the same time, should also allow us to obtain critical information about these structures that have been difficult to study at the moment.

描述(由申请人提供)：虽然是一种相对较新的技术， 原子力显微镜(AFM)已被证明是一种强大的结构工具 生物学。然而，尽管在获取高分辨率地表方面取得了成功 环境条件下多种大分子的结构，原子力显微镜的能力 用于研究大型的、单个的复合体，这些复合体通常 弹性仍然非常有限，主要是因为变形和损坏 由扫描尖端在内部施加相当高的压力引起 针尖和样品之间的接触面积。因此，即使是非常尖锐的 提示：在大型结构上，分辨率相对较低。 为了克服这一问题，扩大原子力显微镜的解释能力 在单分子水平上的分子结构，我们已经开发出 第一台在常温下工作的液氮原子力显微镜 压力(低温原子力显微镜)。我们的结果清楚地表明，在低温条件下 温度下，分子刚性显著提高，允许清晰 以及能够可靠和可重现地获得高分辨率图像。有了这个 新的技术，我们已经能够揭示结构构象 几个以前未知的大分子。 在这一继续申请中，我们寻求继续发展这一点 在结构生物学中应用的新技术，特别是 强调单一、灵活的复合体。我们将构建一个多功能的 将包括一系列样品制备的集成系统 能力。我们还将推动这项技术，以提高其分辨率 保存完好、快速冷冻的标本(在深度蚀刻后)。要实现 这个目标，除了工具的改进之外，我们还计划利用 在我们的低温原子力显微镜中，新开发的纳米管尖端和“单原子”探针。AS 作为这个项目的一个组成部分，我们还将把这个方法应用到几个 精心挑选的问题，包括IgM、蛋白质的结构 分布在细胞膜上，核孔的表面结构 复杂的病毒和一种被包裹的病毒。这些应用程序将提供初始的 方法的验证，但同时也应该允许我们 获取关于这些结构的关键信息，这些结构很难 现在就学习吧。