This thesis describes an investigation of the potential of electron diffraction for studying three dimensional sub-micro-crystals of proteins and pharmaceuticals. A prerequisite for using electron... Show moreThis thesis describes an investigation of the potential of electron diffraction for studying three dimensional sub-micro-crystals of proteins and pharmaceuticals. A prerequisite for using electron diffraction for structural studies is the predictable availability of tiny crystals. A method for growing such crystals using heterogeneous nucleation is demonstrated. The heterogeneous nucleant (in this case hair fibers) was serendipitously selected. Four different proteins (lysozyme, glucose isomerase, a Fab fragment and potato protease inhibitor) were shown to nucleate preferentially on the selected substrate and sub-micro crystals were grown. Further studies on the mechanism of heterogeneous nucleation using lysozyme as a test protein and different imaging techniques such as atomic force and fluorescent microscopy are also discussed. Sub-micron crystals of potato protease inhibitor and lysozyme were subject of electron diffraction studies. A detailed description of the diffraction experiments is presented. A special focus is given on the sample preparation procedure and in particular the vitrification and the cryo-preservation of the crystals. The preliminary results showed that the heterogeneously grown nano-crystals are well ordered and suitable for electron diffraction. The high beam sensitivity of the protein nano-crystals appeared to be the rate limiting step in the data collection, not allowing orientation of the crystals (a technique used in electron diffraction studies of inorganic crystals) or a 3D data collection of a single crystal (a technique used in X-ray protein crystallography). This suggested that new approaches for data collection and data analysis needed to be developed. Optimization of the diffraction data collection, as is described in this thesis allowed high diffraction resolution (up to 2.1_) to be obtained from vitrified lysozyme crystals. An algorithm for unit cell determination of randomly oriented diffraction patterns of different crystals is presented. The method was used for the analysis of the electron diffraction data acquired from lysozyme nano-crystals. The methods for collecting and analyzing electron diffraction data from lysozyme crystals were also confirmed in the case of penicilline type nano-crystals. The motivation behind these studies and the results obtained are discussed. In this case a crystalline powder sample was subjected to electron diffraction studies. Resolutions up to 0.8_ were obtained from oxacillin crystals and up to 1_ from penicillin G crystals. The unit cell parameters found from the analysis of electron diffraction data with the algorithm presented in the previous chapter were consistent with the unit cell parameters obtained by X-ray crystallography on the same two types of penicillin. Show less
