Phasing using high intensity free-electron laser radiation

X-ray free-electron lasers (XFELs) provide extremely bright X-ray pulses of femtosecond duration, that promise to revolutionize structural biology, as they can be used to collect diffraction data from micrometer-sized crystals while outrunning ra- diation damage. The high fluence of the XFEL pulses induces severe electronic radiation damage to the sample, and especially the heavy atoms are strongly ion- ized by the X-ray radiation. The aim of this thesis is to test if it may be possible to use this specific radiation damage effect as a new approach to phasing.By simulating serial femtosecond crystallography experiments at different X-ray fluence conditions, I describe that it is possible to use a Radiation damage-Induced Phasing scheme to retrieve the coordinates of the heavy atoms, and to correctly phase the model structure. Experimental data showed an effective reduction of the scattering power of a heavy atom inside a chemically modified protein, and of the sulfurs in a native protein. From the analysis of these experimental data, quantitative methods have been developed to retrieve information about the effective ionization of the damaged atomic species. The same analysis demonstrated that statistical methods can be used to sort the collected diffraction patterns, according to photon flux impinging on the sample. The knowledge of the real experimental conditions is critical for the success of high intensity phasing technique.