...if, of course, (1) your protein allows to be significantly overexpressed (2) adding inhibitors can protect your protein from proteolysis long enough to collect NMR spectra (3) your protein does not interact with inhibitors or molecules that have significant presence in crude cell-extract...
Automated protein NMR structure determination in crude cell-extract.
Etezady-Esfarjani T, Herrmann T, Horst R, Wuthrich K.
J Biomol NMR. 2006 Jan;34(1):3-11.
Department of Molecular Biology and Joint Center for Structural Genomics, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California, 92037, USA.
touraj@mol.biol.ethz.ch
A fully automated, NOE-based NMR structure determination of a uniformly 13C,15N-labeled protein was achieved in crude cell-extract, without purification of the overexpressed protein. Essentially complete sequence-specific assignments were obtained using triple resonance experiments, based on the high intensity of the resonances from the overexpressed protein relative to those of the background. For the collection of NOE distance constraints, efficient discrimination between NOE cross peaks from the target protein and background signals was achieved using the programs ATNOS and CANDID. In the iterative ATNOS/CANDID procedure, the identification of the desired protein NOEs is initially guided by the self-consistency of the protein NOE-network. Although the intensities of the signals in this network vary over a wide range, and are in many instances comparable to or smaller than those of the background, the first cycle of calculations resulted in the correct global polypeptide fold, and the structure was then refined in six subsequent cycles using the intermediate NMR structures for additional guidance. The experience gained with this work demonstrates that the ATNOS/CANDID procedure for automatic protein structure determination is highly robust and reliable in the presence of intense background signals, and might thus also represent a platform for future protein structure determinations in physiological fluids.
Linear Mode
