Developing novel approaches for time resolved structural biology.

今天结构生物学的技术进步为研究生物分子提供了新的方法。在X射线晶体学实验中，现在更容易研究随时间的变化，例如跟踪配体结合或催化反应。与此同时，进行晶体学实验的方式也发生了变化。一个很好的例子是连续晶体学实验，其中数千个纳米晶体被用来生成复合数据集。同样，这些纳米晶体可用于检索结构变化或随时间观察到的转变的动态信息。巧合的是，生成限定的纳米晶体样品远非容易。我们开发了一种优化晶体生长尺寸和均匀性的工作流程[1]，并在Diamond Light Source I24微焦点光束线和日本XFEL源SACLA上定期进行连续数据收集。您的博士学位致力于优化两个关键目标的纳米结晶：目标1：Hsp 90，一种与维持许多癌症有关的伴侣[2]。可以形成与辅分子伴侣和客户蛋白复合的Hsp 90的非常小的晶体（特别是Hsp 90/cdc 37/Braf复合物）。连续纳米晶体学将使结构确定能够理解Hsp 90如何帮助蛋白质成熟。目标2：Pdx 1，一种治疗疟疾或结核病的药物目标。我们已经描述了由Pdx 1进行的维生素生物合成中的反应中间体[3]。优化该系统的串行数据收集将允许您绘制酶的动态变化，直接观察晶体中的催化循环。与高度创新的CASE合作伙伴道格拉斯仪器[4]合作的微接种方法允许控制每微升的晶体数量和实验体积的缩放。纳米结晶的高通量方法也将使用微流体平台，您将参与南安普顿的开发[5]。

Technical advances in structural biology today allow for new ways to study biomolecules. In the X-ray crystallographic experiment, it is now much easier to study changes over time, for example to follow ligand binding or catalytic reactions. At the same time, the way in which a crystallographic experiment is undertaken changes. A good example is the serial crystallography experiment in which thousands of nanocrystals are used to generate composite datasets. Equally, these nanocrystals can be used to retrieve dynamic information on structural changes, or transitions observed over time. Paradoxically, generating defined nanocrystalline samples is far from easy. We developed a workflow for optimising crystal growth for size and homogeneity [1] and routinely carry out serial data collection at the Diamond Light Source I24 microfocus beamline and at the Japanese XFEL source SACLA. Your PhD engages in optimising nano-crystallisation for two critical targets: Target 1: Hsp90, a chaperone implicated in maintaining many cancers [2]. Very small crystals of Hsp90 in complex with co-chaperones and client proteins can be formed (specifically the Hsp90/cdc37/Braf complex). Serial nano-crystallography will enable structure determination to understand how Hsp90 aids in protein maturation. Target 2: Pdx1, a drug target for malaria or tuberculosis. Reaction intermediates in vitamin biosynthesis carried out by Pdx1 have been described by us [3]. Optimising this system for serial data collection will allow you to map dynamic changes of the enzyme, directly observing the catalytic cycle in crystals. Micro-seeding approaches in collaboration with the highly innovative CASE partner Douglas Instruments [4] allow control of the number of crystals per microliter and scaling of experimental volumes. High-throughput approaches in nano-crystallisation will also use microfluidic platforms that you will be involved in developing at Southampton [5].