About
Integrated Strategy
The five Scientific Cores provide essential intellectual, computational, and experimental infrastructure for development of a general sequence/structure based stategy for functional assignment. While the Scientific Cores are the intellectual and technological center of the EFI, the four Bridging Projects serve to test and calibrate the functional predictions from the Scientific Cores. The Bridging Projects evaluate predicted functions using custom, focused libraries of substrates, products, and analogs to screen for in vitro functions. Such validation is essential to the successful development of a general strategy which will be broadly applicable to proteins from any superfamily thus providing the maximum benefit to the scientific community.
The integrated strategy developed by the EFI for functional assignment of unknown enzymes is summarized below:
1) identify isofunctional families based on sequence relationships (Superfamily/Genome Core)
2) analyze the operon contexts where possible to identify other enzymes that are potentially part of the same metabolic pathway (Superfamily/Genome Core and Bridging Projects)
3) clone, express, and purify targeted enzymes for in vitro studies (Protein Core)
4) structurally characterize purified targets and determine structures of liganded complexes as appropriate (Structure Core)
5) if structures cannot be determined, use homology modeling to obtain high-quality structural templates (Computation Core)
6) use in silico ligand docking to generate rank-ordered lists of predicted substrates (Computation Core)
7) screen purified proteins with chemical libraries for enzymatic activity to determine in vitro function (Bridging Projects)
8) when appropriate, elucidate in vivo function through focused genetics, transcriptomics, and metabolomics or any combination thereof (Microbiology Core)
9) annotation transfer of new function to sufficiently close homologues (Superfamily/Genome Core)
