Investigation of protein structure determination using X-ray free-electron lasers

With the advent of fourth generation radiation sources, X-ray free-electron lasers (XFEL’s), several fields of research, including atomic and molecular sciences, matter at extreme conditions, X-ray imaging, obtained a tool that allows the realization of experiments at conditions and time scales previously inaccessible for researchers. Time scales of atomic motions and extreme temperatures and pressures that occur naturally in the cores of the largest stars became available to the researchers disposal. X-FEL radiation generation is based on the self-amplified spontaneous emissionprinciple (SASE). Timing properties of an X-FEL, combined with femtosecond optical or infra-red laser that is used to trigger specific states in the sample, enabled studies of processes that occur at time scales of atomic motion in a time-resolved manner. One of the promising applications of this new radiation source is the possibility to expand the area of biomolecular structure determination by using intense, ultra-short X-ray pulses [Neu12]. The first step in the direction of single biomolecule imaging was made by retrieving the structure of a known protein arranged in a nanosizedcrystal [Bou12, Cha11]. In this dissertation an exhaustive study about the methodology and capabilities of Serial Femtosecond Crystallography (SFX) for novel protein structure determination is presented. Two unknown protein structures were solved from tens of thousands of in-vivo grown micro-crystals using X-FEL radiation. Comprehensive verification and assessment of SFX for novel protein structure determination from micro-crystals using homologous structures to solve the phase problem is presented and necessary improvements to this method are discussed. Description and influence of the variation of X-ray pulses and protein crystals properties are presented. Possibilities of de-novo protein structure determination using the high intensity multi-wavelength anomalous dispersion (MAD) method [Son11] or by direct phase retrieval using information encoded in shape transforms around Bragg peaks measured from protein nano-crystals [Spe11] are also discussed