Project No. 2160

Primary Supervisor

Dr Ivo Tews – University of Southampton

Co-Supervisor(s)

Dr S. Mark Roe – University of Sussex
Dr Jonathan West – University of Southampton

Industry Partner

Drs Agata Butryn & Pierre Aller – Diamond Light Source

CASE Partner

Patrick Shaw Stewart – Douglas Instruments Ltd

Summary

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].