Three-dimensional (3D) structural determination of biological macromolecules is not only critical to understanding their mechanisms, but also has practical applications. Combining the high resolution imaging of transmission electron microscopy (TEM) and efficient computer processing, protein structures in solution or in two-dimensional (2D) crystals can be determined. The lipid monolayer technique uses the high affinity binding of 6His-tagged proteins to a Ni-nitrilotriacetic (NTA) lipid to create high local protein concentrations, which facilitates 2D crystal formation. In this study, several proteins have been crystallized using this technique, including the SARS virus Nsp15 endonuclease and the human Toll-like receptor (TLR) 3 extracellular domain (ECD). Single particle analysis can determine protein structures in solution without the need for crystals. 3D structures of several protein complexes had been solved by the single particle method, including IniA from Mycobacterium tuberculosis, Nsp15 and TLR3 ECD. Determining the structures of these proteins is an important step toward understanding pathogenic microbes and our immune system.

Thesis--M. Med. Sc., University of Hong Kong, 1982.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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HREM imaging was performed using the Jeol 4000ex microscope on specimens prepared from an as-grown ingot of semi-insulating Gallium Arsenide. Various low angle grain boundaries were imaged in the [110] orientation, misorientations varying between 4°-13°. Detailed study of a grain boundary of 11.5° misorientation about the [110] rotation axis has been carried out. Burgers vector analysis showed the presence of perfect 60° and [001] dislocations. Modelling of the [001] dislocation has been carried out using the Tersoff potential, Bond Order Potential and a tight binding Hamiltonian for GaAs, using Chadi (1984) parameters. The dislocation core was associated with an 8-membered and two 5-membered rings. Assum- ing there is a minimum of wrong bonds, we predict that the core has two wrong bonds, one being Ga-Ga, and the other As-As, both in equivalent positions where the two 5-membered rings were appended to the 8-membered ring. The Ga-Ga bond is considerably shorter and hence stronger than the As-As bond. Band structure calculations performed using a Vogl (1983) sp³s* Hamiltonian revealed deep states in the gap, which are associated with atoms in the core only. Using Stadelmann's (1987) EMS program, successful image matching of calculated images of the [001] dislocation has been achieved with the experimental image, using the atomic structure generated by tight binding relaxation. Ga and As being only two atomic numbers apart have similar scattering factors and cannot be easily distinguished in the experimental image. The equivalence of the position of the two wrong bonds greatly eases image matching as it is no longer necessary to know which is the Ga-Ga , and which is the As-As bond. This is the first suggested model of the [001] dislocation in GaAs, to the best of my knowledge. It is found to be similar to the atomic structure of the 90° partial dislocation in silicon (Bigger et al., 1992). No account of segregation of impurities to the grain boundary, or the [001] dislocation core is taken here, though it is very likely that an impurity atom would sit itself in this large space. The relaxed atomic structure for the 60° dislocation showed a doubling of periodicity along the dislocation line, similar to that found in the 30° partial in Si. The core consists of a 7-membered and a 5-membered ring with a minimum of two wrong bonds. In addition to this, quantitative comparisons of the [001] HREM image and simulated structures have been made and an iterative structure refinement carried out in order to achieve the best image matching. The resultant 'experimental-best-fit' structure was not found to be physically or chemically plausible.