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Type of Document Dissertation Author Bhatia, Smita Author's Email Address smita@sb.fsu.edu URN etd-11162003-234228 Title Combined Crystallographic and Cryo-Electron Microscopic Analysis of Adeno-Associated Virus Type 2 Degree Doctor of Philosophy Department Molecular Biophysics, Institute of Advisory Committee
Advisor Name Title K.A Taylor Committee Member M.S Chapman Committee Member N.L Greenbaum Committee Member R.H Reeves Committee Member T.A Cross Committee Member Keywords
- Molecular Packing
- 3D Reconstruction
- Virus Structure
- Cryo-Em
- AAV-2
Date of Defense 2003-07-15 Availability unrestricted Abstract Adeno-associated virus type-2 (AAV-2) is a leading candidatevector for gene therapy. My PhD is a part of the ongoing research
project towards the long-term goals of engineering AAV to evade
immune neutralization when repeated doses are required, and of
modulating the specificity of cellular targeting.
The specific aim of my PhD research is the structural analysis of
AAV-2 using X-ray crystallography and cryo-Electron Microscopy
(cryo-EM). The two parts of this thesis follow my participation in
the crystallographic structure determination of AAV-2 at 3Å
resolution. In the first part, cryo-EM has been used to visualize
the wild type AAV-2 (wt-AAV2) and its structure determined using
the principles of icosahedral three-dimensional reconstruction. A
difference map has been calculated by subtracting the atomic
structure of the major component, viral protein 3 (VP3), from the
EM density of the wt-AAV2. The results indicate the probable
location of the viral nucleic acid and the other minor viral
proteins, VP1 and VP2, not imaged in the crystallographic
structure. This research lays the foundation for the future
studies of AAV-2-antibody complexes that will be studied by the
same techniques.
The second part of the research involves the analysis of crystal
contacts of the virus in the crystallographic unit cell. Surface
features such as molecular shape and chemical character determine
interactions of a molecule with its surroundings in vivo or
in crystalline forms. Amino acids involved in particle
interactions have been identified from buried surface area
calculations. This research addresses the question of what drives
a virus to choose one packing scheme over another and whether the
crystal contacts carry any biological information. The residues
buried in inter-particle contacts play a stabilizing role in the
packing and are often a part of a surface with demonstrated
biological function such as immunogenic stimulation and receptor
recognition. The results of this research will form the groundwork
for the study of virus-receptor and virus-antibody complexes.
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28.8 Modem 56K Modem ISDN (64 Kb) ISDN (128 Kb) Higher-speed Access 01Title.pdf 17.42 Kb 00:00:04 00:00:02 00:00:02 00:00:01 < 00:00:01 02Committee.pdf 36.72 Kb 00:00:10 00:00:05 00:00:04 00:00:02 < 00:00:01 03Acknowledgements.pdf 49.88 Kb 00:00:13 00:00:07 00:00:06 00:00:03 < 00:00:01 04Contents.pdf 58.35 Kb 00:00:16 00:00:08 00:00:07 00:00:03 < 00:00:01 05Abstract.pdf 55.27 Kb 00:00:15 00:00:07 00:00:06 00:00:03 < 00:00:01 06Chapter1.pdf 11.95 Mb 00:55:20 00:28:27 00:24:54 00:12:27 00:01:03 07Chapter2.pdf 1.78 Mb 00:08:15 00:04:14 00:03:42 00:01:51 00:00:09 08Chapter3.pdf 14.70 Mb 01:08:03 00:34:59 00:30:37 00:15:18 00:01:18 09Chapter4.pdf 46.96 Kb 00:00:13 00:00:06 00:00:05 00:00:02 < 00:00:01 10References.pdf 122.24 Kb 00:00:33 00:00:17 00:00:15 00:00:07 < 00:00:01 11Biosketch.pdf 85.50 Kb 00:00:23 00:00:12 00:00:10 00:00:05 < 00:00:01