Dynamic Structural Biology – from nanocrystals to time-resolution (CASE project)

Primary supervisor:
Dr Ivo Tews, Biological Sciences, University of Southampton
Co-supervisors:
Dr S. Mark Roe, School of Life Sciences, University of Sussex
Dr Jonathan West, Medicine, University of Southampton

Project Summary

Significant technical developments are revolutionising structural biology and change the way in which a crystallographic experiment is approached. Noise-free detection, advances in synchrotron X-ray sources and availability of pulsed X-ray free electron laser sources (XFEL) now firmly establish serial crystallography. Thousands of nanocrystals each contribute a single x-ray exposure. The method is able to add dynamic information on structural changes or transitions, observed over time. Enzymes or higher order molecular complexes are prominent targets.

Micro- or nanocrystalline samples are required to unlock this capability. We developed a workflow for optimising crystal growth for size and homogeneity [1] and demonstrated serial data collection with a fixed target delivery approach using a nano-fabricated chip-based support system, capable of delivering one structure per hour either at the Diamond Light Source (I24 microfocus beamline) or at an XFEL source (SACLA, Japan).

The PhD will optimise nano-crystallisation to study two high value targets. Micro-seeding approaches are developed in collaboration with the highly innovative CASE partner Douglas Instruments [2]. Novel high-throughput approaches in nano-crystallisation using microfluidic platforms are developed at Southampton [3].

Target 1: Hsp90, a chaperone implicated in maintaining many cancers [4]. Very small crystals of Hsp90 in complex with co-chaperones and client proteins can be formed (specifically the Hsp90/cdc37/Braf complex). The size of these crystals is such that they are not suitable for standard data collection, and serial nano-crystallography will enable structure determination to understand how Hsp90 aids in protein maturation.

Target 2: Pdx1, an enzyme that synthesises vitamin B6 from two carbohydrates and ammonia. Crystallisation of several key intermediates is established, and the complex cascade of reactions has been described by us [5]. Serial data collection to map the highly dynamic changes in the enzyme is now required, investigating sugar ring opening, ammonia transfer, and migration of intermediates.