Currently, there are two principal areas of focus in our laboratory. One involves structure determination of membrane proteins at atomic resolution by electron crystallography. Projects underway include crystallization and structure determination of the oxalate carrier OxlT (in collaboration with Prof. Peter Maloney at Johns Hopkins University School of Medicine, and Dr. Jacqueline Milne, NCI), and electron crystallographic analysis of light-induced conformational changes in the proton pump bacteriorhodopsin (in collaboration with Dr. Richard Henderson, MRC Cambridge, England). Over the course of the last year we have initiated systematic efforts to obtain crystals of OxlT suitable for high resolution analysis. We have also determined the structure of the cytoplasmically """"""""open"""""""" conformation of bacteriorhodopsin to a resolution of 3.2 Angstroms, and presented a complete stereochemical mechanism to explain proton pumping by this protein. A second area of research involves structure determination of large protein complexes by analysis of images of single particles obtained by high resolution electron microscopy. Using the 1,800 kD complex of the core domain of pyruvate dehydrogenase as a model molecule, we are establishing methods to carry out such an analysis at high resolution (in collaboration with Dr. Jacqueline Milne, NCI). We have already obtained a three-dimensional reconstruction of the core domain complex at a resolution of about 14 Angstroms and expect to improve it further over the course of the next year.

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010278-03
Application #
6435274
Study Section
(LB)
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2000
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Fenton, Andrew K; Hobley, Laura; Butan, Carmen et al. (2010) A coiled-coil-repeat protein 'Ccrp' in Bdellovibrio bacteriovorus prevents cellular indentation, but is not essential for vibroid cell morphology. FEMS Microbiol Lett 313:89-95
Milne, Jacqueline L S; Subramaniam, Sriram (2009) Cryo-electron tomography of bacteria: progress, challenges and future prospects. Nat Rev Microbiol 7:666-75
Lengyel, Jeffrey S; Milne, Jacqueline L S; Subramaniam, Sriram (2008) Electron tomography in nanoparticle imaging and analysis. Nanomedicine (Lond) 3:125-31
Bartesaghi, A; Sprechmann, P; Liu, J et al. (2008) Classification and 3D averaging with missing wedge correction in biological electron tomography. J Struct Biol 162:436-50
Narasimha, Rajesh; Aganj, Iman; Bennett, Adam E et al. (2008) Evaluation of denoising algorithms for biological electron tomography. J Struct Biol 164:7-17
Lefman, Jonathan; Morrison, Robert; Subramaniam, Sriram (2007) Automated 100-position specimen loader and image acquisition system for transmission electron microscopy. J Struct Biol 158:318-26
Subramaniam, Sriram; Bartesaghi, Alberto; Liu, Jun et al. (2007) Electron tomography of viruses. Curr Opin Struct Biol 17:596-602
Heymann, Jurgen A W; Hayles, Mike; Gestmann, Ingo et al. (2006) Site-specific 3D imaging of cells and tissues with a dual beam microscope. J Struct Biol 155:63-73
Subramaniam, Sriram (2006) The SIV surface spike imaged by electron tomography: one leg or three? PLoS Pathog 2:e91
Milne, Jacqueline L S; Wu, Xiongwu; Borgnia, Mario J et al. (2006) Molecular structure of a 9-MDa icosahedral pyruvate dehydrogenase subcomplex containing the E2 and E3 enzymes using cryoelectron microscopy. J Biol Chem 281:4364-70

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