Investigation Into The Three-Dimensional Structure Of Periplaneta Fuliginosa Densovirus

For a long time people have been trying to understand the relationship between the structure and function of the macromolecular complexes. In general, only with a good understanding of the structure of the macromolecular complexes at atomic resolution level, can its function be better understood. Therefore, investigations into the three-dimensional (3D) virus structures at a high resolution are crucial to the understanding of the fundamental processes of virus assembly and cellular events during viral infections.X-ray crystallography was the first method for structural determination of macromolecular complexes. It can be used to study atomic structure of 3-D crystals. The key requirement for a successful X-ray analysis is that the sample studied must grow to well-ordered 3-D crystals. However, due to this requirement, it is difficult to apply the technique of the X-ray crystallography in structural studies of viruses.Nuclear magnetic resonance (NMR) spectroscopy is a promising tool for studies of the atomic structure of small molecules and the macromolecule in solution. A unique feature of the technique is its ability to probe the dynamics of molecular conformations. However, the interpretation of the complex spectra obtained from the macromolecule with big molecular weight is exceptionally difficult. Therefore, NMR spectroscopy are normally used to study the structures of relatively small molecules with the molecular weight less than 35 Kda.Electron cryomicroscopy (cryoEM) is a fast emerging technique for three-dimensional structural reconstruction of macromolecular complexes. Through using this technique, images of frozen-hydrated macromolecules can be obtained by quickly freezing the specimen to the liquid nitrogen or liquid helium temperature, and then keeping the temperature below -150 C during the course ofimaging. CryoEM images are recorded using a low electron dose (<20e / A2/s) to minimize the radiation damage to the specimen. This allows investigation into three-dimensional structures of macromolecular complexes at a high resolution. In the conventional electron microscopy, the stabilization of the sample in microscope vacuum is achieved through negative stain, chemical fixation, and dehydration, etc. This results in the loss of the high-resolution structural information of the biological sample. Therefore, the conventional methods can only be used for studies at a very low-resolution level. In contrast, in cryoEM , the cooling of hydrated specimen is exceptionally fast, avoiding the formation of crystalline ice. It is just like that the whole sample is embedded in a vitreous ice with the depth of about 2um. The layer vitreous ice can not only prop up the sample as support membrane, but also preserve the water in frozen-hydrated sample in the high vacuum of the microscope, giving a better protection of the sample of hydrated macromolecular complexes. As a result, the sample can be placed in or near to in the physiological active state, and the high resolution structure information of the sample can be retained. This makes possible the 3D structure study of macromolecular complexes at a high resolution.Parvoviruses are able to infect human and mammalian cells, and to cause a lot of diseases. Many efforts have been made to study the biological characteristics of the parvoviruses. The biological characteristic of many vertebrate parvoviruses have been clarified, for example the genome organization, the structure of capsids protein of virus particle, and the relationship between the structure and its function. In particular, among these studies, the crystal structure of three vertebrate parvoviruses, canine parvovirus(CPV), feline panleukopenia virus(FPV), and minute virus of mice(MVM), have been determined at near-atomic resolution using the technique of X-ray crystallography. However, the studies of densoviruses are still limited. This thesis will contribute to this subject through investigation into the 3D structure of Periplaneta fuliginosa densovirus (pfDNV).Cockroaches, an ancient insect...