X-ray crystallography is a powerful method in determining the structure of both small molecules and macromolecules and is now routinely applied in many scientific fields. However, to apply this method, there is an unavoidable problem to tackle: the Phase Problem, which arises because the phases of a scattered x-ray cannot be measured in diffraction experiment and the original structure cannot be retrieved only with the measurable amplitudes. This thesis presents two approaches in the development of macromolecular phasing methods. One approach presented here utilizes molecular envelope of NMR structures for molecular replacement (MR) phasing with the program FSEARCH at low resolution (about 6 Å). X-ray crystallography and NMR are complementary tools in structural biology. However, it is often difficult to use NMR structures as search models in MR to phase crystallographic data. For this purpose, in our study, several targets with both crystallographic and NMR structures available have been tested. The test protocol involves four steps: (1) Model preparation, NMR structures were processed into averaged polyalanine model, and centroid NMR models have also been tested; (2) Six-dimensional low resolution search were carried out by FSEARCH to find the best match between observed and calculated structure factors; (3) Apply the solution (4) Model building and refinement. In our tests, FSEARCH was able to find the correct translation and orientation of the search model in the crystallographic unit cell, while conventional MR procedures were unsuccessful. The other approach presented in this thesis is protein complex structure completion using IPCAS (Iterative Protein Crystal structure Automatic Solution). Protein complexes have been concerned as essential components in almost every cellular process. X-ray crystallography method is quite useful in studying the nature of protein complexes. In this study, we demonstrated a protein complex completion procedure from a partial molecular replacement (MR) solution using IPCAS. IPCAS is a direct-method aided dual-space iterative phasing and model-building procedure. The test cases were carefully selected from a practical perspective and IPCAS could build the whole complex from one or less than one subunit once molecular replacement method could give a partial solution. Before delivering to IPCAS, MR solution model examination and improvement might be necessary. The IPCAS iteration procedure involves (1) real-space model building and refinement; (2) direct-method aided reciprocal-space phase refinement; and (3) phase improvement through density modification. In our tests, IPCAS is able to extend the full length complex from a less than 30% starting model while conventional model building procedure were unsuccessful. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

A model has been developed for calculating x-ray line emission from spherical plasmas. The main features of this method are: (1) Plasma parameters are obtained from a one-dimensional Lagrangian hydrodynamics and heat flow code. (2) Multi-frequency groups: the line structure can be reproduced with the desired accuracy by adjusting the number of frequency groups. (3) Self consistent, time dependent excited level populations and radiation fluxes: the code starts with coronal populations, calculates the ensuing radiation flux and then recalculates the populations and so on, iterating until convergence is reached. (4) Goemetrical groups of rays groups by spherical impact parameters. (5) Line broadening due to ionic thermal agitation and Doppler shift due to the net plasma flow velocity. Inclusion of the flow velocity shift would be different without the multi-frequency group treatment. The method has been applied to an aluminum target, and the results are in good agreement with previous experimental work. The total energy, summed over all lines, as well as the line intensity ratios (which are a sensitive measure of agreement with experiment) were predicted with good accuracy. The pictures that would be seen by a pinhole camera are also calculated by the code.

This thesis presents work on theoretical tools used to study transient and quantum-fluctuating impurity potentials that arise in resonant x-ray scattering and ultracold atomic gases. These tools fall under two main classes, functional determinants for exact evaluation of many-fermion matrix elements, and the variational polaron transformation. The following work carefully introduces both approaches and compares theoretical predictions to known experimental and computational results. In several cases this thesis presents arguments that experiments on high-temperature superconducting cup rates must be reinterpreted in terms of a quasiparticle picture. Where no experimental data exist, predictions are made and suggestions given for new uses for simple experimental techniques. For example, indirect resonant inelastic x-ray scattering turns out to be a versatile pseudo gap probe, and radio frequency absorption of a fermi gas with an impurity can detect a repulsively-bound state. / Physics

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Cellulosomes are the most efficient plant cell wall degradation machines discovered to date. All cellulosomal components contain protein modules connected by linkers of varying lengths, which are predicted to be flexible. Consequently, structural studies of the cellulosome have employed a “dissect and build” strategy, whereby individual modules are studied in isolation with the hope to later model the intact complex. However, representative individual structures are now available for all of the cellulosome modules and many questions still remain. The studies described in this thesis depart from the ‘dissection’ stage and enter the ‘build’ stage of cellulosome structural studies of the cellulosome from Clostridium thermocellum. We first describe the crystal structure of a heterodimeric complex comprising the type-II cohesin (CohII) from cell surface anchoring protein SdbA and a trimodular C-terminal truncation of the CipA scaffoldin protein containing the ninth type-I cohesin module (CohI9), a linker, the X-module (X), and the type-II dockerin module (DocII). This structure revealed novel intertwining of scaffoldin molecules and extensive reciprocal contacts between the CohI9 and the X-module of another scaffoldin molecule. Sedimentation velocity experiments indicate dimerization also occurs in solution. We have carried out the crystallization and structural analysis of a heterotrimeric complex containing the CohI9—X-DocII:CohII complex bound to the type-I dockerin module (DocI) from the Cel9D enzyme, which represents the largest cellulosome fragment ever determined. Identical inter-scaffoldin interactions were observed in the heterotrimeric complex structure as were seen in the heterodimeric complex. However, small angle X-ray scattering (SAXS) studies indicate that this dimerization does not occur in solution. The crystal structures and additional SAXS studies reveal flexibility in the CohI9—X linker that is surprisingly restricted to two dimensions. In addition, this structure provides the first evidence of an orientation bias in DocI binding. Finally, SAXS was used to investigate modular orientations and linker flexibility in several cellulosome fragments. These studies indicate that cellulosomal linkers exhibit restricted and in some cases highly restrained flexibility. Specifically, scaffoldin linkers display two dimensional motions, enzymes maintain close contact with their cognate DocI modules, and enzyme positions rotate about 90° relative to neighbouring enzymes on the scaffoldin. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2010-09-02 02:50:11.12

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