Understanding the rules of sample preparation for single particle cryo-EM

了解单颗粒冷冻电镜的样品制备规则

• 批准号: BB/X007227/1
• 负责人: Stephen Muench
• 金额: $ 78.42万
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX007227%2F1
• 关键词: Understanding; rules; sample; preparation; single

Structural biology is an important area of science. By understanding the structure of a protein or protein complexes we can start to understand how it works and how mutations cause disease. Furthermore, by understanding a proteins structure we can start to design new therapeutics that can modulate its function to treat different disease states. However, obtaining a structure is not easy, proteins are very small, dynamic and delicate. One of the main techniques which has grown significantly in recent years is cryo electron microscopy cryo-EM which allows you to see proteins at atomic resolution where we can see the position of individual atoms. Whilst the microscopes and the way in which we process the data are well developed there is a current limitation in our understanding of how we prepare the sample for the microscope. This preparation involves freezing the protein in a very thin "film" of buffer very quickly to create vitreous ice. Although current approaches work well for some proteins, for many proteins it creates problems that can result in the protein being damaged or fully unfolded. Moreover, the protein can adopt a preferred orientation such that only one view is seen. This can result in artefacts in the structure or often an inability to solve the structure. Many of these problems result from interactions with the interface between the buffer and air (air water interface (AWI)). Our groups have already started to understand some of the affects that can happen, often in a time dependant manner but this is just the tip of the iceberg. This grant will allow us to better understand what happens during sample preparation by looking at how proteins behave (1) over time from sub ms to seconds, (2) on different grids and environments used to support the sample and (3) with different additives added that can change the behaviour of the sample such as detergents to lower surface tension. For our first objective we have a unique piece of equipment that will allow us to make grids from sub ms (not possible to our knowledge by any other group) to seconds. This unique capability coupled with our access to world class EM facilities means we can for the first time look at how the protein behaves over time during the sample preparation process and see how we might minimise some of the negative effects. For the second objective we will see how the chemistry of the support film of the grid which holds the sample and the environmental gas used changes the protein behaviour. We already have pilot data to show a big difference between gold and copper grids indicating that the chemistry of the grid may influence the protein. The third objective will look at how different additives such as detergents influence protein behaviour, we have seen for different proteins that they change their behaviour in the presence of additives and these can make the difference between success and failure but how they work is poorly understood. By better understanding this we may be able to recommend specific additives for different problems during sample preparation. Together this will allow us to better define the rules that affect sample preparation making these experiments more successful to improve data collection and make certain proteins tractable to structure determination.

结构生物学是一个重要的科学领域。通过了解蛋白质或蛋白质复合物的结构，我们可以开始了解它是如何工作的，以及突变如何导致疾病。此外，通过了解蛋白质结构，我们可以开始设计新的治疗方法，可以调节其功能以治疗不同的疾病状态。然而，获得结构并不容易，蛋白质非常小，动态和微妙。近年来发展迅速的主要技术之一是冷冻电子显微镜冷冻EM，它可以让你看到原子分辨率的蛋白质，我们可以看到单个原子的位置。虽然显微镜和我们处理数据的方式已经发展得很好，但目前我们对如何为显微镜准备样品的理解存在局限性。这种制备包括将蛋白质冷冻在非常薄的缓冲液“膜”中，以非常快地产生玻璃状冰。虽然目前的方法对某些蛋白质效果很好，但对许多蛋白质来说，它会产生问题，导致蛋白质被损坏或完全展开。此外，蛋白质可以采用优选的取向，使得仅看到一个视图。这可能导致结构中的伪影或通常无法解决结构。这些问题中的许多是由缓冲液和空气之间的界面（空气-水界面（AWI））的相互作用引起的。我们的团队已经开始了解可能发生的一些影响，通常是以时间依赖的方式，但这只是冰山一角。这项资助将使我们能够更好地了解在样品制备过程中发生了什么，通过观察蛋白质的行为（1）随着时间的推移，从亚毫秒到秒，（2）在用于支持样品的不同网格和环境中，以及（3）添加不同的添加剂，可以改变样品的行为，如洗涤剂，以降低表面张力。对于我们的第一个目标，我们有一个独特的设备，这将使我们能够使网格从亚毫秒（不可能由我们的知识，任何其他组）秒。这种独特的能力加上我们拥有世界一流的EM设施，这意味着我们可以第一次看到蛋白质在样品制备过程中随着时间的推移如何表现，并了解我们如何最大限度地减少一些负面影响。对于第二个目标，我们将看到保持样品的网格的支撑膜的化学性质和所使用的环境气体如何改变蛋白质的行为。我们已经有试验数据显示，金和铜网格之间存在很大差异，表明网格的化学性质可能会影响蛋白质。第三个目标将研究不同的添加剂如洗涤剂如何影响蛋白质的行为，我们已经看到不同的蛋白质在添加剂的存在下改变了它们的行为，这些可以决定成功和失败，但它们是如何工作的知之甚少。通过更好地了解这一点，我们可以针对样品制备过程中的不同问题推荐特定的添加剂。总之，这将使我们能够更好地定义影响样品制备的规则，使这些实验更成功地改善数据收集，并使某些蛋白质易于结构测定。