The availability of extremely bright X-ray sources such as the recently developed hard X-ray Free Electron Laser (XFEL) at Stanford are creating entirely new fields of research across all major disciplines. Femtosecond nanocrystallography is rapidly maturing into a major new technique for structural biologists, enabling high-resolution structural data to be collected from nanocrystals which are too small to measure using conventional X-ray sources. This has enabled a significant decrease in time for structural retrieval of hard to crystallise proteins. The need to understand how to conduct these types of experiments, however, is becoming ever more apparent in the scientific community. A more detailed understanding of the process from nanocrystal sample preparation to serial femtosecond nanocrystallography experiments and structure analysis will be presented here.

The results presented will be based on imaging and diffraction experiments carried out at both LCLS-XFEL and third generation synchrotrons. We find that the XFEL data contains evidence of both electronic and structural disorder whilst the synchrotron data provides a window into the elastic strain properties of protein crystals that can influence the structure retrieval. We show that applying this additional information to both the crystallographic analysis and XFEL analysis can improve the structure, helping to ameliorate the effects of having imperfect crystals.