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Time-resolved methodologies to provide both spatial and temporal resolution in Electron Microscopy

在电子显微镜中提供空间和时间分辨率的时间分辨方法

基本信息

批准号：
BB/P026397/1
负责人：
Stephen Muench
金额：
$ 16.94万
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：
2017
资助国家：
英国
起止时间：
2017 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FP026397%2F1
关键词：
Time resolved methodologies provide both

项目摘要

Structural biology has been key to unlocking the secrets of mutational change within proteins and provides the platform for therapeutic intervention. Traditionally, X-ray crystallography has provided the high resolution information required, but it is dependent on well-ordered crystals, limiting its scope, especially for membrane proteins and large protein complexes. Yet these systems demand structural investigation. Alternatively, electron microscopy (EM) is ideally suited to studying protein complexes and membrane proteins but has traditionally been hindered by a more modest resolution. However, EM has undergone a "resolution revolution" driven by more stable microscopes, advanced processing algorithms, rapid automatic data collection and new direct electron detectors which offer greater quality images. As a consequence atomic resolution of non-symmetric proteins is becoming the norm and previously intractable but important medical targets are now amenable to structure based drug design using EM derived structures. A powerful but under exploited advantage of EM is the ability to trap different conformational states that exist within the sample. However, this is currently reliant on computational sorting of different conformational states that exist within the sample in the "ground state". To overcome this, time-resolved methodologies can be used. This methodology relies on mixing a protein and substrate rapidly before freezing in a liquid cryogen to trap the reaction intermediate in the ms time frame. The significant challenge is that to image a protein with the electron microscope it must be frozen on a specialised grid in vitreous ice which is thin enough for the electrons to pass through but not thinner than the sample being studied. This is commonly achieved using an automated "blotting" device (such as the Vitrobot), which blots away excess solution from the grid leaving a suitable amount such that upon rapid freezing a vitreous ice layer is formed of an appropriate thickness. Our primary route for time-resolved studies will be to rapidly mix protein and substrate in a microfluidic chamber before directly spraying it onto a grid and plunge freezing. We have a basic setup that is capable of a time resolution >50ms but methodologies are still in their infancy and have yet to realise their full potential. This is limited by two major factors, the first is in obtaining a suitable ice thickness with inconsistencies of the direct spraying approach. By screening a range of grid types including the recently developed micro-fibre grids that have been designed to negate this problem we are confident we can find a working solution. The second limitation is in obtaining a reliable, reproducible and fast rapid mixing device. Through a new collaboration with Hamburg University we will translate the technology designed for cutting edge diffraction technologies (x-ray free electron laser and SAXS) to develop a rapid mixing unit. By integrating with the leading grid preparation system (Vitrobot) we can develop a universal system accessible by any interested EM group, moving this technology from niche to mainstream. This funding will allow us to take the first significant step towards this by generating a prototype system capable of producing time resolution >5ms in a more controlled and reliable manner. Looking forward the next significant milestone in structural biology will be the capture of temporal as well as spatial resolution, greatly increasing our understanding of proteins and protein complexes and moving us away from the "static" view that currently predominates in the field.

结构生物学一直是解开蛋白质内突变变化秘密的关键，并为治疗干预提供了平台。传统上，X射线晶体学提供了所需的高分辨率信息，但它依赖于有序的晶体，限制了其范围，特别是对于膜蛋白和大蛋白复合物。然而，这些系统需要结构性的研究。另外，电子显微镜（EM）非常适合研究蛋白质复合物和膜蛋白，但传统上受到更适度分辨率的阻碍。然而，EM已经经历了一场“分辨率革命”，这是由更稳定的显微镜、先进的处理算法、快速自动数据收集和提供更高质量图像的新的直接电子探测器驱动的。因此，非对称蛋白质的原子分辨率正在成为常态，并且以前难以处理但重要的医学靶点现在适合于使用EM衍生结构的基于结构的药物设计。EM的一个强大但未充分利用的优势是捕获样品中存在的不同构象状态的能力。然而，这是目前依赖于不同的构象状态，存在于样品中的“基态”的计算排序。为了克服这一点，可以使用时间分辨方法。该方法依赖于在液体冷冻剂中冷冻之前快速混合蛋白质和底物，以在ms时间范围内捕获反应中间体。重要的挑战是，要用电子显微镜对蛋白质进行成像，必须将其冷冻在玻璃冰中的专用网格上，玻璃冰足够薄，可以让电子通过，但不能比所研究的样品薄。这通常使用自动化的“吸除”装置（例如Vitrobot）来实现，其从网格中吸除过量的溶液，留下合适的量，使得在快速冷冻时形成具有适当厚度的玻璃状冰层。我们进行时间分辨研究的主要途径是在微流控室中快速混合蛋白质和底物，然后将其直接喷射到网格上并进行冷冻。我们有一个基本的设置，能够实现> 50毫秒的时间分辨率，但方法仍处于起步阶段，尚未实现其全部潜力。这受到两个主要因素的限制，第一个是在获得合适的冰厚度与直接喷洒方法的不一致性。通过筛选一系列网格类型，包括最近开发的微纤维网格，这些网格旨在消除这个问题，我们相信我们可以找到一个有效的解决方案。第二个限制是获得可靠的、可再现的和快速的快速混合装置。通过与汉堡大学的新合作，我们将把为尖端衍射技术（X射线自由电子激光和SAXS）设计的技术转化为快速混合装置。通过与领先的网格准备系统（Vitrobot）集成，我们可以开发一个通用系统，任何感兴趣的EM组都可以使用，将这项技术从利基市场推向主流。这笔资金将使我们能够通过生成能够以更可控和可靠的方式产生时间分辨率>5ms的原型系统来迈出重要的第一步。展望未来，结构生物学的下一个重要里程碑将是捕获时间和空间分辨率，大大增加我们对蛋白质和蛋白质复合物的理解，并使我们远离目前在该领域占主导地位的“静态”观点。

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

On-grid and in-flow mixing for time-resolved cryo-EM.

DOI：
10.1107/s2059798321008810
发表时间：
2021-10-01
期刊：
Acta crystallographica. Section D, Structural biology
影响因子：
0
作者：
Klebl DP;White HD;Sobott F;Muench SP
通讯作者：
Muench SP

Need for Speed: Examining Protein Behavior during CryoEM Grid Preparation at Different Timescales.

DOI：
10.1016/j.str.2020.07.018
发表时间：
2020-11-03
期刊：
Structure (London, England : 1993)
影响因子：
0
作者：
Klebl DP;Gravett MSC;Kontziampasis D;Wright DJ;Bon RS;Monteiro DCF;Trebbin M;Sobott F;White HD;Darrow MC;Thompson RF;Muench SP
通讯作者：
Muench SP

Towards sub-millisecond cryo-EM grid preparation.

DOI：
10.1039/d2fd00079b
发表时间：
2022-11-08
期刊：
FARADAY DISCUSSIONS
影响因子：
3.4
作者：
Klebl, David P.;Kay, Robert W.;Sobott, Frank;Kapur, Nikil;Muench, Stephen P.
通讯作者：
Muench, Stephen P.

Cryo-EM structure of human mitochondrial HSPD1.