Illuminated in Time : Evaluating a Reporter System and a Molecular Cage for Time- Resolved Serial Femtosecond X-ray Crystallography at the European X-ray Free- Electron Laser

Abstract

The invention of X-ray free-electron lasers revolutionised the field of time-resolved crystallography by utilising the ultrashort X-ray pulses to probe reactions down to the femtosecond timescale, permitting unprecedented insights into the dynamics of biological processes. At the European X-ray Free-Electron Laser, the megahertz repetition rate and high peak brilliance enable the collection of multiple time points within a single experiment at a high spatio-temporal resolution. However, beamtime is highly limited and requires extensive preparatory work and additional data-processing steps compared to traditional crystallography. In this study, the preparatory work and data analysis are represented by analysing two distinct systems that both aim to enhance experiments performed at the Single Particles, Clusters, and Biomolecules & Serial Femtosecond Crystallography instrument at the European XFEL facility. Initial experiments on thymine dimerisation in DNA oligomers to validate the instrumental set-up for pump-probed experiments involved UV irradiation, subsequent testing for structural changes, as well as crystallisation and collection of diffraction data at a synchrotron source. The analysis of time-resolved serial femtosecond crystallography diffraction data was performed as part of an experiment on the applicability of an oxygen photocage by processing the diffraction patterns and refining the structures. While leakage from the photocage led to premature oxygenation of the test molecule, which could not be sufficiently compensated by a reduced contact time, the DNA reporter system showed structural changes upon UV irradiation in the initial tests and, with further improvement, could be implemented as a validation system improving the efficient use of beamtime.