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Breaking the Cage: Transformative Time-resolved Crystallography using Fixed Targets at Synchrotrons and XFELs

打破牢笼：在同步加速器和 XFEL 上使用固定目标的变革性时间分辨晶体学

基本信息

批准号：
BB/W001950/1
负责人：
Jonathan Worrall
金额：
$ 56.85万
依托单位：
University of Essex
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2022
资助国家：
英国
起止时间：
2022 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FW001950%2F1
关键词：
Breaking Cage Transformative Time resolved

项目摘要

Efforts to understand how enzyme catalysis and protein-ligand binding work (or how they fail to work properly) is a vital research field in the life sciences. The knowledge gained has significance for the development of efficient biosynthetic materials, for applications in the biochemical, biotechnology and industrial sectors and in developing new medicines. Obtaining the structures of enzymes is an essential part of this effort. X-ray crystallography is a technique that reveals the individual atoms that make up an enzyme molecule, showing how they are joined to each other to form larger 3D structures. These structures and how they change over time as an enzyme performs its work are key factors for our understanding of enzyme function. This 'time-resolved' aspect is analogous to moving from a single photograph to a movie, and is tremendously powerful but also very challenging to achieve. To uncover this detailed 3D arrangement of atoms within an enzyme, powerful X-ray sources, called synchrotron storage rings and X-ray free electron lasers (XFELs), are used to visualise crystallised versions of the enzyme, using micron sized X-ray beams. The structure of the enzyme can then be deduced from the way that the X-rays are 'diffracted' as they pass through the crystal. The crystals that are grown in the laboratory for these experiments, including many important pharmaceutical targets for therapies, are very often only available as tiny, micron-sized (one-thousandth of a millimetre) 'microcrystals'. We will use silicon 'chips' for efficient delivery to the X-ray beam, for optimum high-throughput and high 'hit rate' (a 'hit' is when the micron size X-ray beam is diffracted by a microcrystal). A chip is a device containing thousands of shaped wells each of which can trap a microcrystal. Each chip can hold up to 25,600 microcrystals and each well, with its trapped microcrystal, is exposed in turn to the X-rays beam at the selected experimental facility used (the Diamond synchrotron in the UK or SACLA in Japan). The different facilities used have very different properties so can be used to provide complementary information by for example probing different timescales.The X-ray diffraction patterns gathered from all the microcrystal hits are combined and analysed to create a 3D model (structure) of the enzyme. Using chips for sample delivery we can obtain a complete 3D structure of an enzyme in less than one hour, which is a very efficient use of these expensive X-ray facilities. We will first study enzymes in their 'resting' states before they undergo catalysis (do their work), giving us the initial structures of the enzymes. We will use oxygen and nitric oxide and photocages to initiate catalysis or ligand binding in microcrystals. Photocages are compounds that contain a trapped molecule of interest, which is held securely and only released by a brief flash of a laser beam. By collecting diffraction patterns from the microcrystals at varying time delays after the laser flash we can capture time dependent changes and build up an accurate molecular movie of the enzyme in action. Crucially, we are able to produce these structures at room temperature, close to the conditions under which enzymes function in nature. This allows their dynamic movements related to their function to be followed much more accurately compared to the cryogenic conditions (liquid nitrogen temperature, approximately -173 oC) at which crystal structures are typically produced. Our approach allows us to follow reactions over a wide timescale from microseconds to seconds or minutes, building up a complete picture of catalysis.

努力了解酶催化和蛋白质配体结合如何工作（或它们如何不能正常工作）是生命科学的一个重要研究领域。所获得的知识对于开发高效的生物合成材料、在生化、生物技术和工业部门的应用以及开发新药具有重要意义。获得酶的结构是这项工作的重要组成部分。x射线晶体学是一种揭示构成酶分子的单个原子的技术，显示它们如何相互连接以形成更大的3D结构。这些结构以及它们在酶发挥作用时如何随时间变化是我们理解酶功能的关键因素。这种“时间分辨”方面类似于从一张照片移动到一部电影，它非常强大，但实现起来也非常具有挑战性。为了揭示酶内原子的这种详细的3D排列，使用了强大的x射线源，称为同步加速器存储环和x射线自由电子激光器（XFELs），使用微米大小的x射线束来可视化酶的结晶版本。酶的结构可以从x射线穿过晶体时的“衍射”方式推断出来。这些实验中在实验室中生长的晶体，包括许多重要的治疗药物靶点，通常只能以微小的微米大小（千分之一毫米）的“微晶体”获得。我们将使用硅“芯片”来有效地输送到x射线束，以获得最佳的高通量和高“命中率”（“命中率”是指微米大小的x射线束被微晶体衍射时）。芯片是一种包含数千个形状阱的装置，每个阱都可以捕获一个微晶体。每个芯片最多可容纳25,600个微晶体，每个阱及其捕获的微晶体依次暴露在选定的实验设备（英国的Diamond同步加速器或日本的SACLA）的x射线束中。所使用的不同设备具有非常不同的特性，因此可以通过例如探测不同的时间尺度来提供补充信息。从所有微晶命中收集的x射线衍射图被组合和分析，以创建酶的3D模型（结构）。使用芯片进行样品递送，我们可以在不到一个小时的时间内获得酶的完整3D结构，这是对这些昂贵的x射线设备的非常有效的利用。我们将首先研究酶在进行催化（完成它们的工作）之前的“静息”状态，给我们酶的初始结构。我们将使用氧气和一氧化氮和光笼来启动微晶体中的催化或配体结合。光笼是一种含有被捕获的感兴趣分子的化合物，它被牢牢地抓住，只有在激光束的短暂闪光下才能释放出来。通过收集微晶体在激光照射后不同时间延迟的衍射图，我们可以捕捉到与时间相关的变化，并建立起起作用的酶的精确分子膜。至关重要的是，我们能够在室温下产生这些结构，接近酶在自然界中发挥作用的条件。这使得与晶体结构通常产生的低温条件（液氮温度，约-173℃）相比，可以更准确地跟踪与其功能相关的动态运动。我们的方法使我们能够在从微秒到秒或分钟的广泛时间尺度上跟踪反应，从而建立起催化的完整图景。