Automated protein NMR structure determination in crude cell-extract

Journal of Biomolecular NMR
Touraj Etezady-EsfarjaniKurt Wüthrich

Abstract

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...Continue Reading

References

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Jun 24, 2004·Journal of Biomolecular NMR·Touraj Etezady-EsfarjaniKurt Wüthrich
Jul 21, 2004·Journal of Structural and Functional Genomics·Wolfgang PetiKurt Wüthrich
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Citations

Dec 31, 2008·Biochemistry·Gary J PielakImola G Zigoneanu
Jul 16, 2008·PloS One·David S Burz, Alexander Shekhtman
May 16, 2007·Journal of Structural Biology·Philipp Selenko, Gerhard Wagner
Jul 11, 2006·Magnetic Resonance in Chemistry : MRC·Touraj Etezady-EsfarjaniKurt Wüthrich

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