Developing novel approaches for time resolved structural biology.

当今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射线结晶学实验中，现在研究随时间的变化要容易得多，例如跟踪配体结合或催化反应。与此同时，进行结晶学实验的方式也发生了变化。一个很好的例子是系列结晶学实验，在这个实验中，数千个纳米晶体被用来生成复合数据集。同样，这些纳米晶体可以用来检索关于结构变化或随时间观察到的转变的动态信息。矛盾的是，制造出特定的纳米晶体样品远非易事。我们开发了一套针对尺寸和均匀性优化晶体生长的工作流程[1]，并定期在钻石光源I24微焦光束线和日本XFEL光源SACLA进行连续数据采集。您的博士致力于优化两个关键目标的纳米结晶：目标1：HSP90，一种与许多癌症的维持有关的伴侣[2]。可以形成非常小的Hsp90晶体，与辅助伴侣和客户蛋白形成复合体(特别是Hsp90/cdc37/BRAF复合体)。连续纳米结晶学将使结构测定能够理解Hsp90如何帮助蛋白质成熟。目标2：治疗疟疾或结核病的药物靶点PDX1。我们已经描述了Pdx1进行的维生素生物合成的反应中间体[3]。对该系统进行系列数据收集的优化将使您能够绘制酶的动态变化图，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]合作的微种方法允许控制每微升晶体的数量和实验体积的缩放。纳米结晶化的高通量方法还将使用您将在南安普敦参与开发的微流体平台[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].