21007SP Anaerobic methods in serial protein crystallography

The field of structural biology is currently undergoing a rapid development. Serial crystallography has opened up many new opportunities as it enables to record multiple diffraction patterns from a continuous supply of protein microcrystals. The advantages of serial approaches enable us to a) collect data on crystals that are otherwise too small, b) limit the effects of radiation damage by spreading the applied dose, c) provide the opportunity to derive structural information under physiological conditions, and d) provide an opportunity for time-resolved experiments at synchrotrons and X‐ray free electron lasers (XFELs). Sample preparation and presentation impose unique challenges on serial experiments and are one of the factors that limit effectiveness of data collection. This can be particularly challenging for biological systems that require very specific working conditions, i.e. are temperature, oxygen or light sensitive.

As part of the ongoing research efforts, XFEL Hub together with the University of Oxford and the I24 team have been working on the development of a generalizable strategy in which serial data could be collected from protein microcrystals under anaerobic conditions. In our recent study (Rabe, P. et al., IUCrJ, 2020, 7, DOI: 10.1107/S2052252520010374), we have presented a new method based on the fixed target mount developed by the I24 team. To provide optimal, oxygen-free environment, we developed a way to prepare samples under anaerobic conditions in holders that were modified to minimize air penetration. We further validated this modified design by developing a fluorescence-based assay and directly demonstrated its application on three different biological systems.

As our design does not offer perfect impermeability, in the future we would like to introduce further modifications to our system which would aim at improving gas penetration properties. The role of the student will be to characterize the new designs. At the beginning the student would learn how to operate the main equipment to be used (anaerobic chambers and spectrophotometer) and how to prepare, handle and load the samples into the standard and anaerobic fixed target holders. The next step of the project would include assembly of different mounts and sealing material suggested by the supervisor and the student in the anaerobic chamber. The student would test gas impermeability of the mounts and sealing material in spectroscopic measurements using an oxygen-sensitive fluorescent compound. In the next step the different mounts and sealing materials would be examined with samples provided by the collaborators within the XFEL Hub BAG time, during X-ray data collection.

At the end of the placement the student will be asked to give a presentation and provide the final report in a written form to the supervisor.