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Protein structure determination in living cells by in-cell NMR spectroscopy


Investigating proteins ‘at work’ in a living environment at atomic resolution is a major goal of molecular biology, which has not been achieved even though methods for the three-dimensional (3D) structure determination of purified proteins in single crystals or in solution are widely used. Recent developments in NMR hardware and methodology have enabled the measurement of high-resolution heteronuclear multi-dimensional NMR spectra of macromolecules in living cells (in-cell NMR)1,2,3,4,5. Various intracellular events such as conformational changes, dynamics and binding events have been investigated by this method. However, the low sensitivity and the short lifetime of the samples have so far prevented the acquisition of sufficient structural information to determine protein structures by in-cell NMR. Here we show the first, to our knowledge, 3D protein structure calculated exclusively on the basis of information obtained in living cells. The structure of the putative heavy-metal binding protein TTHA1718 from Thermus thermophilus HB8 overexpressed in Escherichia coli cells was solved by in-cell NMR. Rapid measurement of the 3D NMR spectra by nonlinear sampling of the indirectly acquired dimensions was used to overcome problems caused by the instability and low sensitivity of living E. coli samples. Almost all of the expected backbone NMR resonances and most of the side-chain NMR resonances were observed and assigned, enabling high quality (0.96 ångström backbone root mean squared deviation) structures to be calculated that are very similar to the in vitro structure of TTHA1718 determined independently. The in-cell NMR approach can thus provide accurate high-resolution structures of proteins in living environments.

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Figure 1: Stability of E. coli cells expressing TTHA1718 under NMR measurement conditions.
Figure 2: Rapid acquisition of 3D NMR spectra of TTHA1718 in living E. coli cells.
Figure 3: Collection of NOE-derived distance restraints in TTHA1718 in living E. coli cells.
Figure 4: NMR solution structure of TTHA1718 in living E. coli cells.

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Protein Data Bank

Data deposits

Atomic coordinates of the structures of TTHA1718 in E. coli cells and in vitro have been deposited in the Protein Data Bank under accession codes 2ROG and 2ROE, respectively. Chemical shifts have been deposited in the BioMagResBank under accession numbers 11037 and 11035.


  1. Serber, Z. et al. High-resolution macromolecular NMR spectroscopy inside living cells. J. Am. Chem. Soc. 123, 2446–2447 (2001)

    CAS  Article  Google Scholar 

  2. Serber, Z., Corsini, L., Durst, F. & Dötsch, V. In-cell NMR spectroscopy. Methods Enzymol. 394, 17–41 (2005)

    CAS  Article  Google Scholar 

  3. Serber, Z. et al. Investigating macromolecules inside cultured and injected cells by in-cell NMR spectroscopy. Nature Protocols 1, 2701–2709 (2006)

    CAS  Article  Google Scholar 

  4. Reckel, S., Hänsel, R., Löhr, F. & Dötsch, V. In-cell NMR spectroscopy. Prog. Nucl. Magn. Reson. Spectrosc. 51, 91–101 (2007)

    CAS  Article  Google Scholar 

  5. Selenko, P. & Wagner, G. Looking into live cells with in-cell NMR spectroscopy. J. Struct. Biol. 158, 244–253 (2007)

    CAS  Article  Google Scholar 

  6. Ellis, R. J. Macromolecular crowding: obvious but underappreciated. Trends Biochem. Sci. 26, 597–604 (2001)

    CAS  Article  Google Scholar 

  7. Burz, D. S., Dutta, K., Cowburn, D. & Shekhtman, A. Mapping structural interactions using in-cell NMR spectroscopy (STINT-NMR). Nature Methods 3, 91–93 (2006)

    CAS  Article  Google Scholar 

  8. McNulty, B. C., Young, G. B. & Pielak, G. J. Macromolecular crowding in the Escherichia coli periplasm maintains α-synuclein disorder. J. Mol. Biol. 355, 893–897 (2006)

    CAS  Article  Google Scholar 

  9. Dedmon, M. M., Patel, C. N., Young, G. B. & Pielak, G. J. FlgM gains structure in living cells. Proc. Natl Acad. Sci. USA 99, 12681–12684 (2002)

    ADS  CAS  Article  Google Scholar 

  10. Selenko, P., Serber, Z., Gade, B., Ruderman, J. & Wagner, G. Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes. Proc. Natl Acad. Sci. USA 103, 11904–11909 (2006)

    ADS  CAS  Article  Google Scholar 

  11. Sakai, T. et al. In-cell NMR spectroscopy of proteins inside Xenopus laevis oocytes. J. Biomol. NMR 36, 179–188 (2006)

    CAS  Article  Google Scholar 

  12. Inomata, K. et al. High-resolution multi-dimensional NMR spectroscopy of proteins in living human cells. Nature 10.1038/nature07839 (this issue)

  13. Wüthrich, K. NMR of Proteins and Nucleic Acids (Wiley, 1986)

    Book  Google Scholar 

  14. Reardon, P. N. & Spicer, L. D. Multidimensional NMR spectroscopy for protein characterization and assignment inside cells. J. Am. Chem. Soc. 127, 10848–10849 (2005)

    CAS  Article  Google Scholar 

  15. Serber, Z., Ledwidge, R., Miller, S. M. & Dötsch, V. Evaluation of parameters critical to observing proteins inside living Escherichia coli by in-cell NMR spectroscopy. J. Am. Chem. Soc. 123, 8895–8901 (2001)

    CAS  Article  Google Scholar 

  16. Barna, J. C. J., Laue, E. D., Mayger, M. R., Skilling, J. & Worrall, S. J. P. Exponential sampling, an alternative method for sampling in two-dimensional NMR experiments. J. Magn. Reson. 73, 69–77 (1987)

    ADS  CAS  Google Scholar 

  17. Schmieder, P., Stern, A. S., Wagner, G. & Hoch, J. C. Improved resolution in triple-resonance spectra by nonlinear sampling in the constant-time domain. J. Biomol. NMR 4, 483–490 (1994)

    CAS  Article  Google Scholar 

  18. Rovnyak, D. et al. Accelerated acquisition of high resolution triple-resonance spectra using non-uniform sampling and maximum entropy reconstruction. J. Magn. Reson. 170, 15–21 (2004)

    ADS  CAS  Article  Google Scholar 

  19. Mueller, G. A. et al. Global folds of proteins with low densities of NOEs using residual dipolar couplings: application to the 370-residue maltodextrin-binding protein. J. Mol. Biol. 300, 197–212 (2000)

    CAS  Article  Google Scholar 

  20. Serber, Z. et al. Methyl groups as probes for proteins and complexes in in-cell NMR experiments. J. Am. Chem. Soc. 126, 7119–7125 (2004)

    CAS  Article  Google Scholar 

  21. Rosen, M. K. et al. Selective methyl group protonation of perdeuterated proteins. J. Mol. Biol. 263, 627–636 (1996)

    CAS  Article  Google Scholar 

  22. Güntert, P., Mumenthaler, C. & Wüthrich, K. Torsion angle dynamics for NMR structure calculation with the new program DYANA. J. Mol. Biol. 273, 283–298 (1997)

    Article  Google Scholar 

  23. Kainosho, M. et al. Optimal isotope labelling for NMR protein structure determinations. Nature 440, 52–57 (2006)

    ADS  CAS  Article  Google Scholar 

  24. Li, C. et al. Differential dynamical effects of macromolecular crowding on an intrinsically disordered protein and a globular protein: implications for in-cell NMR spectroscopy. J. Am. Chem. Soc. 130, 6310–6311 (2008)

    CAS  Article  Google Scholar 

  25. Kraulis, P. J., Domaille, P. J., Campbell-Burk, S. L., Van Aken, T. & Laue, E. D. Solution structure and dynamics of Ras p21-GDP determined by heteronuclear three- and four-dimensional NMR spectroscopy. Biochemistry 33, 3515–3531 (1994)

    CAS  Article  Google Scholar 

  26. Laue, E. D., Mayger, M. R., Skilling, J. & Staunton, J. Reconstruction of phase sensitive 2D NMR spectra by maximum entropy. J. Magn. Reson. 68, 14–29 (1986)

    ADS  CAS  Google Scholar 

  27. Herrmann, T., Güntert, P. & Wüthrich, K. Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA. J. Mol. Biol. 319, 209–227 (2002)

    CAS  Article  Google Scholar 

  28. Cornilescu, G., Delaglio, F. & Bax, A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR 13, 289–302 (1999)

    CAS  Article  Google Scholar 

  29. Cornell, W. D. et al. A second generation force field for the simulation of proteins, nucleic acids, and organic molecules. J. Am. Chem. Soc. 117, 5179–5197 (1995)

    CAS  Article  Google Scholar 

  30. Koradi, R., Billeter, M. & Güntert, P. Point-centered domain decomposition for parallel molecular dynamics simulation. Comput. Phys. Commun. 124, 139–147 (2000)

    ADS  CAS  Article  Google Scholar 

  31. Gardy, J. L. et al. PSORT-B: Improving protein subcellular localization prediction for gram-negative bacteria. Nucleic Acids Res. 31, 3613–3617 (2003)

    CAS  Article  Google Scholar 

  32. Gardy, J. L. et al. PSORTb v.2.0: expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis. Bioinformatics 21, 617–623 (2005)

    CAS  Article  Google Scholar 

  33. Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10, 1–6 (1997)

    CAS  Article  Google Scholar 

  34. Bendtsen, J. D., Nielsen, H., von Heijne, G. & Brunak, S. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340, 783–795 (2004)

    Article  Google Scholar 

  35. Thorstenson, Y. R., Zhang, Y., Olson, P. S. & Mascarenhas, D. Leaderless polypeptides efficiently extracted from whole cells by osmotic shock. J. Bacteriol. 179, 5333–5339 (1997)

    CAS  Article  Google Scholar 

  36. Hayashi, N., Matsubara, M., Takasaki, A., Titani, K. & Taniguchi, H. An expression system of rat calmodulin using T7 phage promoter in Escherichia coli. Protein Expr. Purif. 12, 25–28 (1998)

    CAS  Article  Google Scholar 

  37. Kraulis, P. J. ANSIG: a program for the assignment of protein 1H 2D NMR spectra by interactive computer graphics. J. Magn. Reson. 84, 627–633 (1989)

    ADS  CAS  Google Scholar 

  38. Güntert, P. Automated NMR protein structure calculation. Prog. Nucl. Magn. Reson. Spectrosc. 43, 105–125 (2003)

    Article  Google Scholar 

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The authors thank S. Kuramitsu for providing the plasmid encoding TTHA1718, and D. Nietlispach for setting up 3D NMR experiments with nonlinear sampling schemes and 15N relaxation experiments, T. Anzai for assistance with NMR measurements, and H. Koyama and A. Iwasaki for sample preparations. This work was supported in part by CREST, Japan Science and Technology Agency (JST), the Molecular Ensemble Program, RIKEN, Grants-in-Aid for Scientific Research of Priority Areas from the Japanese Ministry of Education, Sports, Culture, Science, and Technology on ‘Molecular Soft Interactions Regulating Membrane Interface of Biological Systems’ and ‘Molecular Science for Supra Functional Systems – Development of Advanced Methods for Exploring Elementary Process’, and by the Volkswagen Foundation.

Author Contributions B.O.S., M.S., P.G. and Y.I. designed the research and wrote the manuscript. D.S., A.S. and T.I. conducted the research including sample preparation, data acquisition, resonance assignment and structure calculation. M.M. and M.W. helped with NMR measurements. M.M. prepared TTHA1718 mutants. J.H. and T.H. measured NMR data on TTHA1718 mutants and 15N-relaxation experiments. N.H. provided the expression vector for calmodulin. M.Y. measured NMR data on calmodulin in living E. coli cells. T.M. helped during the preparation and characterisation of TTHA1718.

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Correspondence to Yutaka Ito.

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Sakakibara, D., Sasaki, A., Ikeya, T. et al. Protein structure determination in living cells by in-cell NMR spectroscopy. Nature 458, 102–105 (2009).

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