X-ray analysis of biological macromolecules

The work described in this thesis relates to two topics: the crystallography of the enzyme andalpha;-glucan phosphorylase extracted from rabbit muscle, and the development of a photographic data collection system using a rotation camera. Crystals of phosphorylase in the a and b forms were obtained with the spacegroup P21. These crystals have the phosphorylase tetramer, molecular weight 400,000 daltons, as the asymmetric unit and, therefore, the structure determination using them presents a considerable crystallographic problem. The crystals were also grown in the presence of methyl mercury chloride thereby producing a potential heavy atom derivative. A small amount of work was carried out on these crystals before another crystal form of phosphorylase b was discovered. This second crystal form has the space group P41212 (or P43212) and has the phosphorylase monomer, molecular weight 100,000 daltons, as the asymmetric unit. Although the unit cell volume of these tetragonal crystals is slightly larger than that of the monoclinic crystals, the spacegroup symmetry reduces the number of unique reflections by a factor of approximately 4; hence these crystals are potentially more useful for a structure determination. The tetragonal crystals grow most readily in the presence of inosine monophosphate, and will not grow at all in the presence of adenosine monophosphate. A search for isomorphous heavy atom derivatives of these tetragonal crystals has been conducted with a limited amount of success. Diffusion of platinum, in the form of the platinum diamino-dichloride ion, into the native crystals yields a single site derivative of high occupancy. Various methods of data collection were considered before 6andAring; data sets were collected on the four circle diffractometer for the native and 4 potential heavy atom derivatives. The method of data collection and data processing are discussed in this thesis and the results given. A rotation camera had been built in the laboratory by Mr.andnbsp;M.andnbsp;Pickford and Mr.andnbsp;J.andnbsp;Marsh to a design by Dr.andnbsp;D.I.andnbsp;Brown. The camera was intended for use with crystals which had large unit cells. Testing and further development of this camera was carried out, and is described in this thesis. The films from the rotation camera are processed using a Photoscan P1000 Microdensitometer which produces digital images of the films on magnetic tapes. The development of computer programmes to predict the positions of reflections on rotation films and to calculate their integrated intensities using the microdensitometer output tapes is described. An outline of rotation camera geometry is also given. A series of rotation photographs was. taken with a crystal of lysozyme on the camera in order to check the computer pro- grammes. The first results of these checks were very encouraging. Some initial experiments using a tetragonal phosphorylase b crystal on the camera were carried out with encouraging results.