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.

结构生物学是科学的重要领域。通过了解蛋白质或蛋白质复合物的结构，我们可以开始了解其起作用以及突变如何引起疾病。此外，通过了解蛋白质结构，我们可以开始设计新的治疗剂，以调节其功能以治疗不同的疾病状态。但是，获得结构并不容易，蛋白质非常小，动态且精致。近年来，主要发展的主要技术之一是冷冻电子显微镜冷冻EM，它使您可以在原子分辨率下看到蛋白质，我们可以看到各个原子的位置。尽管显微镜和我们处理数据的方式良好，但是当我们对如何为显微镜准备样本的理解中存在一个局限性。这种制剂涉及在非常薄的缓冲液中冻结蛋白质，以产生玻璃冰。尽管当前的方法适用于某些蛋白质，但对于许多蛋白质，它会产生可能导致蛋白质受损或完全展开的问题。此外，该蛋白质可以采用首选方向，因此只能看到一种观点。这可能导致结构中的人工制品，或者通常无法解决结构。这些问题中有许多是由于与缓冲区和空气之间的界面相互作用（空气水接口（AWI））引起的。我们的小组已经开始理解可能发生的某些影响，通常是在时间依赖的方式上，但这只是冰山一角。这笔赠款将使我们能够通过查看蛋白质的行为（1）如何从子MS到秒，（2）在用于支持样品的不同网格和环境上的表现（1），以及（3）添加了不同的添加剂，可以改变样品的行为，例如添加样品的行为，例如降低表面张力。对于我们的第一个目标，我们拥有独特的设备，可以使我们从Sub MS（据我们所知）到几秒钟的网格。这种独特的能力加上我们获得世界一流的设施的访问意味着我们首次可以研究蛋白质在样本制备过程中随时间的行为，并查看我们如何最大程度地减少某些负面影响。对于第二个目标，我们将看到持有样品和使用环境气体的网格支持膜的化学性能如何改变蛋白质的行为。我们已经有了试验数据，可以显示黄金和铜网格之间的巨大差异，表明网格的化学可能会影响蛋白质。第三个目标将研究不同的添加剂（例如洗涤剂）如何影响蛋白质行为，我们已经看到不同蛋白质在添加剂的存在下改变其行为，这些可以使成功与失败之间有所不同，但是它们的工作方式知之甚少。通过更好地理解这一点，我们可以在样本准备过程中为不同问题推荐特定的添加剂。这将使我们能够更好地定义影响样品制备的规则，从而使这些实验更加成功，以改善数据收集并使某些蛋白质可用于结构确定。