Femtosecond x-ray protein nanocrystallography and correlated fluctuation small-angle x-ray scattering
With the advent of the X-ray free-electron laser (XFEL), an opportunity has arisen to break the nexus between radiation dose and spatial resolution in diffractive imaging, by outrunning radiation damage altogether when using single X-ray pulses so brief that they terminate before atomic motion commences. This dissertation concerns the application of XFELs to biomolecular imaging in an effort to overcome the severe challenges associated with radiation damage and macroscopic protein crystal growth. The method of femtosecond protein nanocrystallography (fsPNX) is investigated, and a new method for extracting crystallographic structure factors is demonstrated on simulated data and on the first experimental fsPNX data obtained at an XFEL. Errors are assessed based on standard metrics familiar to the crystallography community. It is shown that resulting structure factors match the quality of those measured conventionally, at least to 9 angstrom resolution. A new method for ab-initio phasing of coherently-illuminated nanocrystals is then demonstrated on simulated data. The method of correlated fluctuation small-angle X-ray scattering (CFSAXS) is also investigated as an alternative route to biomolecular structure determination, without the use of crystals. It is demonstrated that, for a constrained two-dimensional geometry, a projection image of a single particle can be formed, ab-initio and without modeling parameters, from measured diffracted intensity correlations arising from disordered ensembles of identical particles illuminated simultaneously. The method is demonstrated experimentally, based on soft X-ray diffraction from disordered but identical nanoparticles, providing the first experimental proof-of-principle result. Finally, the fundamental limitations of CFSAXS is investigated through both theory and simulations. It is found that the signal-to-noise ratio (SNR) for CFSAXS data is essentially independent of the number of particles exposed in each diffraction pattern. The dependence of SNR on particle size and resolution is considered, and realistic estimates are made (with the inclusion of solvent scatter) of the SNR for protein solution scattering experiments utilizing an XFEL source.