Methods for initiating and following substrate driven reactions in macromolecular crystallography

The project will exploit serial synchrotron crystallography (SSX) to probe non-reversible reactions in proteins. Typically, crystallography provides a single static snapshot of a molecule. By delivering substrate to a protein crystal and then collecting data at a series of time delays afterwards, a series of snapshots are obtained and a molecular stop motion movie of the protein in action can be formed. Experiments of this type are challenging however as substrate must be delivered in a reproducible manner to large numbers of well characterised crystals and, furthermore, diffusion rates can vary from sample-to-sample and substrate-to-substrate. In this project, a collaboration between I24 and the XFEL-Hub, the student will develop methods for mixing ligand/substrate with protein microcrystals for time-resolved SSX at I24 and beyond.  

Diffusion of the substrate is a limiting factor when driving reactions in protein crystals as during soaking substrate will take some time to reach all molecules in the crystal. As the average enzyme catalytic turnover time is about 60ms, it is important to work with micron-size crystals so that substrate can fully diffuse into the crystal lattice on timescales significantly faster than this. The project will exploit acoustic drop ejection to efficiently and reproducibly deliver small volumes (~100 pL) of substrate to thousands of microcrystals which will be delivered to the X-ray beam using the serial techniques available at Diamond. Diffraction data will be collected at room temperature at time intervals ranging from tens of milliseconds to a few seconds after reaction initiation. The student will make use of standard crystals to characterise hardware and methods, to determine in crystallo reaction rates and identify and resolve limitations of the approach before application to challenging ‘real world’ samples (available through our existing external collaborators) with the ultimate goal of turning drop-on-crystal into a tool available for the Diamond MX user community.