CRANK is a suite that links different macromolecular X-ray crystallographic programs to solve macromolecular crystal structures automatically from experimental phasing data. In chapter 2, several... Show moreCRANK is a suite that links different macromolecular X-ray crystallographic programs to solve macromolecular crystal structures automatically from experimental phasing data. In chapter 2, several new algorithms implemented within CRANK increase the robustness and speed of the structure solution process. The new MULTICOMB program, discussed in chapter 3, provides a new phase combination algorithm for the density modification step of the structure solution process. MULTICOMB implements a novel advanced multivariate function that considers the single-wavelength anomalous diffraction (SAD) data directly, accounts for the correlation between the initial and density-modified maps and refines errors that can occur in a SAD experiment. Testing of these new algorithms with over 100 real data sets showed a dramatic improvement over state-of-the-art methods. These novel methods were also applied in solving the new structure of the DNA-binding protein Sso10a2 from Sulfolobus solfataricus reported in chapter 4. This structure provides insight to the observed differences in behaviour between Sso10a2 and its close homolog Sso10a. The last chapter of this work describes the crystallization conditions for a recombinant, fully glycosylated form of the human C1 inhibitor protein, which is involved in hereditary angioedema, a potentially life threatening condition. Show less
Genomic DNA in bacteria exists in a condensed state, which exhibits different biochemical and biophysical properties from a dilute solution. DNA was concentrated on streptavidin-covered single... Show moreGenomic DNA in bacteria exists in a condensed state, which exhibits different biochemical and biophysical properties from a dilute solution. DNA was concentrated on streptavidin-covered single-walled carbon nanotubes (Strep-SWNTs) through biotin-streptavidin interactions. We reasoned that confining DNA within a defined space through mechanical constraints, rather than by manipulating buffer conditions, would more closely resemble physiological conditions. By ensuring a high streptavidin loading on SWNTs of about 1 streptavidin tetramer per 4 nm of SWNT, we were able to achieve dense DNA binding. DNA is bound to Strep-SWNTs at a tunable density and up to as high as 0.5 mg mL(-1) in solution and 29 mg mL(-1) on a 2D surface. This platform allows us to observe the aggregation behavior of DNA at high concentrations and the counteracting effects of HU protein (a histone-like protein from Escherichia coli strain U93) on the DNA aggregates. This provides an in vitro model for studying DNA-DNA and DNA-protein interactions at a high DNA concentration. Show less
