High-Throughput Structure Determination of Proteins and Protein Complexes
EMSL Project ID
35412
Abstract
The Northeast Structural Genomics Consortium (NESGC, www.nesg.org) is one of four large protein structure production centers supported by the NIH Protein Structure Initiative II (the PSI II). The NESGC used EMSL's High Field NMR facility extensively in 2006, 2007, and 2008 within science theme proposal 19837, entitled Protein Interactions and Interfaces. During this three-year period, NMR data was collected for more than 50 proteins. High resolution structures of many of these are now deposited in the Protein Data Bank and can be seen in the structure gallery at the above web site. We now present a proposal addressing the Biological Interactions and Dynamics science theme that will build on previous achievements in high-throughput NMR data collection and structure determination and investigation of protein-protein and protein-ligand complexes. While continuing to collect NMR data enabling structure determination of about 15 proteins per year for the NESGC, we will place significant emphasis on evaluation and application of a new approach for structure determination of larger proteins and protein complexes. This approach combines sparse experimental restraints such as NOE distances and residual dipolar couplings (RDCs) and alternative labeling strategies with backbone chemical shift assisted computational structure prediction (CS-Rosetta) to achieve accurate, high throughput structure determination of proteins in the 16-25 kDa range. We also plan to conduct CS-Rosetta structure determination without sparse restraints on 15 additional small proteins that are lower priority targets due to higher sequence similarity to proteins that already have structures. To accomplish the proposed work, we are requesting time on EMSL 600, 750, and 800 MHz NMR spectrometers for protein NMR data collection, access to the Chinook supercomputer for CS-Rosetta calculations, and use of EMSL wet lab resources for sample manipulation and other routine work associated with biomolecular NMR spectroscopy. All structures determined will be deposited in the Protein Data Bank and published as appropriate. Many of these proteins come from organisms of interest to DOE, including Shewanella oneidensis, Desulfovibrio vulgaris, and Rhodobacter sphaeroides. Frequently, structures lead to development of functional hypotheses and become the focus of other research projects, sometimes with the assistance of NESGC scientists, other times without such help. In this way, structural genomics is an enabling resource for biology. Furthermore, establishment of CS-Rosetta combined with sparse restraints as a means of routine structure determination of larger proteins would be a significant development in the field.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2009-10-01
End Date
2012-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
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