Journal of Biomolecular NMR

, Volume 34, Issue 1, pp 3–11 | Cite as

Automated Protein NMR Structure Determination in Crude Cell-Extract

  • Touraj Etezady-Esfarjani
  • Torsten Herrmann
  • Reto Horst
  • Kurt Wüthrich
Article

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 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.

Keywords

automated protein NMR structure determination with ATNOS and CANDID network anchoring structural proteomics Thermotoga maritima 

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References

  1. Almeida, F.C., Amorim, G.C., Moreau, V.H., Sousa, V.O., Creazola, A.T., Americo, T.A., Pais, A.P., Leite, A., Netto, L.E., Giordano, R.J., Valente, A.P. 2001J. Magn. Reson.148142146CrossRefADSGoogle Scholar
  2. Bartels, C., Xia, T.H., Billeter, M., Güntert, P., Wüthrich, K. 1995J. Biomol. NMR6110CrossRefGoogle Scholar
  3. Cornell, W.D., Cieplak, P., Bayly, C.I., Gould, I.R., Merz, K.M., Ferguson, D.M., Spellmeyer, D.C., Fox, T., Caldwell, J.W., Kollman, P.A. 1995J. Am. Chem. Soc.11751795197CrossRefGoogle Scholar
  4. Etezady-Esfarjani, T., Herrmann, T., Peti, W., Klock, H.E., Lesley, S.A., Wüthrich, K. 2004J. Biomol. NMR29403406Google Scholar
  5. Galvao-Botton, L.M., Katsuyama, A.M., Guzzo, C.R., Almeida, F.C., Farah, C.S., Valente, A.P. 2003FEBS Lett.552207213Google Scholar
  6. Gronenborn, A.M., Clore, G.M. 1996Protein Sci.5174177Google Scholar
  7. Güntert, P., Dötsch, V., Wider, G., Wüthrich, K. 1992J. Biomol. NMR2619629CrossRefGoogle Scholar
  8. Güntert, P., Mumenthaler, C., Wüthrich, K. 1997J. Mol. Biol.273283298CrossRefGoogle Scholar
  9. Herrmann, T., Güntert, P., Wüthrich, K. 2002aJ. Mol. Biol.319209227CrossRefGoogle Scholar
  10. Herrmann, T., Güntert, P., Wüthrich, K. 2002bJ. Biomol. NMR24171189CrossRefGoogle Scholar
  11. Hubbard, J.A., MacLachlan, L.K., King, G.W., Jones, J.J., Fosberry, A.P. 2003Mol. Microbiol.4911911200CrossRefGoogle Scholar
  12. Koradi, R., Billeter, M., Güntert, P. 2000Comput. Phys. Commun.124139147ADSGoogle Scholar
  13. Lee, K.M., Androphy, E.J., Baleja, J.D. 1995J. Biomol. NMR59396Google Scholar
  14. Luginbühl, P., Güntert, P., Billeter, M., Wüthrich, K. 1996J. Biomol. NMR8136146CrossRefGoogle Scholar
  15. Luginbühl, P., Szyperski, T., Wüthrich, K. 1995J. Magn. Reson. B109229233Google Scholar
  16. Marley, J., Lu, M., Bracken, C. 2001J. Biomol. NMR2071CrossRefGoogle Scholar
  17. Muhandiram, D.R., Farrow, N.A., Xu, G.Y., Smallcombe, S.H., Kay, L.E. 1993J. Magn. Reson.102317321Google Scholar
  18. Nilges, M. 1997Fold. Des.2S53S57CrossRefGoogle Scholar
  19. Oldfield, C.J., Ulrich, E.L., Cheng, Y., Dunker, A.K., Markley, J.L. 2005Proteins59444453CrossRefGoogle Scholar
  20. Ou, H.D., Lai, H.C., Serber, Z., Dötsch, V. 2001J. Biomol. NMR21269273CrossRefGoogle Scholar
  21. Page, R., Peti, W., Wilson, I.A., Stevens, R.C., Wüthrich, K. 2005Proc. Natl. Acad. Sci. USA10219011905CrossRefADSGoogle Scholar
  22. Peti, W., Etezady-Esfarjani, T., Herrmann, T., Klock, H.E., Lesley, S.A., Wüthrich, K. 2004J. Struct. Funct. Genomics5205215CrossRefGoogle Scholar
  23. Sattler, M., Schleucher, J., Griesinger, C. 1999Progr. Nucl. Magn. Reson. Spect.3493158Google Scholar
  24. Serber, Z., Dötsch, V. 2001Biochemistry401431714323CrossRefGoogle Scholar
  25. Spera, S., Bax, A. 1991J. Am. Chem. Soc.11354905492CrossRefGoogle Scholar
  26. Wüthrich, K. 1986NMR of Proteins and Nucleic AcidsWileyNew YorkGoogle Scholar
  27. Yee, A., Chang, X., Pineda-Lucena, A., Wu, B., Semesi, A., Le, B., Ramelot, T., Lee, G.M., Bhattacharyya, S., Gutierrez, P., Denisov, A., Lee, C.H., Cort, J.R., Kozlov, G., Liao, J., Finak, G., Chen, L., Wishart, D., Lee, W., McIntosh, L.P., Gehring, K., Kennedy, M.A., Edwards, A.M., Arrowsmith, C.H. 2002Proc. Natl. Acad. Sci. USA9918251830ADSGoogle Scholar
  28. Zuiderweg, E.R.P., Fesik, S.W. 1989Biochemistry2823872391Google Scholar

Copyright information

© Springer 2006

Authors and Affiliations

  • Touraj Etezady-Esfarjani
    • 1
    • 2
  • Torsten Herrmann
    • 1
    • 2
  • Reto Horst
    • 1
  • Kurt Wüthrich
    • 1
  1. 1.Department of Molecular Biology and Joint Center for Structural GenomicsThe Scripps Research InstituteCaliforniaUSA
  2. 2.Institut für Molekularbiologie und BiophysikETH ZürichSwitzerland

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