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BioinformaticsTarget SelectionNYCOMPS targets are subdivided into three main categories: Pipeline, Nominated and Biological Theme targets. Pipeline targets are selected by a protocol that currently begins with E. coli proteins. These E. coli seeds are predicted membrane proteins that have been expressed successfully in a previous large-scale experiment on membrane proteins carried out by the lab of Gunnar von Heijne (Daley et al. Science. 2005 308:1321-3). We expand these seeds and into 92 prokaryotic genomes (reagent genomes) from which we clone. Nominated targets are seeds selected by individual NYCOMPS experimental groups and expanded by the bioinformatics unit into the 92 reagent genomes. Biological Theme targets are proteins of exceptional biological interest that are cloned by the individual NYCOMPS experimental groups. Data analysisData from experimental trials (cloning, expression, purification, crystallization) are processed and analyzed with the aim to iteratively improve our target selection strategy. Homology ModelingProteins for which the structures have been experimentally determined by NYCOMPS are used as templates for building homology models for other proteins within the same structural family. Protein ProductionNYCOMPS uses high-throughput automated methods to clone, express and assess the suitability for structural studies of hundreds of full-length integral membrane proteins per week (TargetDB). To accomplish this we have built a highly automated pipeline that makes use of several novel robotic processes developed at NYCOMPS. Innovations in automation technology include the first 96 well bacterial membrane isolation technique and methods to rapidly assay the stability of target proteins in a number of different detergents. X-ray CrystallographyNYCOMPS uses state of the art nano-volume crystallization robots to produce membrane protein crystals. NYCOMPS is standardized on high-throughput sitting drop screening methods, but we are investigating HT crystallization under oil, crystallization in lipidic cubic phases and 2D crystallization screening by automated electron microscopy.
NMR SpectroscopyNMR has successfully complemented crystallography for structural studies of water-soluble proteins of moderate size that either do not crystallize, or do not diffract well. The same should hold true for membrane proteins, where crystallization is one of the main challenges to structural study. Thus, the overall goal of the NYCOMPS NMR effort is to develop and implement a robust and relatively rapid set of NMR experiments and analysis procedures to determine the structures of membrane proteins, and apply these methods to NYCOMPS targets. NMR targets, currently less than 150 residues in length, are evaluated in a standard series of detergents by both gel filtration and 2D proton-nitrogen correlation spectra. Standard TROSY-based triple resonance NMR approaches are used as the initial path to obtaining resonance assignments and constraints (phi-psi angles, NOEs, hydrogen bonds, and orientational constraints) for structure determination. To improve throughput and better resolve typically congested membrane protein NMR spectra, G-matrix FT NMR methods for assignments and structural constraints are being implemented, along with automated analysis tools for both GFT and traditional data sets. Oligomeric membrane proteins reconstituted into native lipid bilayers are being approached by solid state NMR. |
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