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- Creators: Alford, Terry
Description
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.
ContributorsKirian, Richard A (Author) / Spence, John C. H. (Committee member) / Doak, R. Bruce (Committee member) / Weierstall, Uwe (Committee member) / Bennett, Peter (Committee member) / Treacy, Michael M. J. (Committee member) / Arizona State University (Publisher)
Created2011
Description
Protein crystallization is a technique for the formation of three-dimensional protein crystals, which is widely utilized by scientists, engineers, and researchers. Protein crystallography allows for protein structures and functions to be studied. As proteins play a central role in biological systems and life itself, a deeper understanding of their structure-function properties is crucial to elucidating fundamental behaviors, such as protein folding in addition to the role that they play in emerging fields, such as, tissue engineering with application to the emerging field of regenerative medicine. However, a significant limitation toward achieving further advancements in this field is that in order to determine detailed structure of proteins from protein crystals, high-quality and larger size protein crystals are needed. Because it is difficult to produce adequate size, high-quality crystals, it remains difficult to determine the structure of many proteins. However, a new method using a microgravity environment to crystallize proteins has proven effective through various studies conducted on the International Space Station (ISS). In the presence of microgravity, free convection is essentially absent in the bulk solution where crystallization occurs, thus allowing for purely random Brownian motion to exist which favors the nucleation and growth of high-quality protein crystals. Many studies from the ISS to date have demonstrated that growing protein crystals in a microgravity environment produces larger and higher-quality crystals. This method provides new opportunities for better structure identification and analysis of proteins. Although there remains many more limitations and challenges in the field, microgravity protein crystallization holds many opportunities for the future of biotechnology and scientific development. The objective of this thesis was to study the crystallization of hen egg white lysozyme (HEWL) and determine the effects of both unit and microgravity on growth/size and quality of HEWL. Through preliminary trials using a universal ground-based reduced-gravity system, the crystallization of HEWL in a simulated microgravity environment was successfully conducted and the results reported are promising. The utility of continuous, scalable ground-based, microgravity platforms for studies on a wide range of material systems and behavior, such as, protein crystallization, has significant implications regarding its impact on many industries, including drug development as well as regenerative medicine.
ContributorsTran, Amanda Marie (Author) / Pizziconi, Vincent (Thesis director) / Alford, Terry (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2020-12