Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Probing a cell-embedded megadalton protein complex by DNP-supported solid-state NMR


Studying biomolecules at atomic resolution in their native environment is the ultimate aim of structural biology. We investigated the bacterial type IV secretion system core complex (T4SScc) by cellular dynamic nuclear polarization–based solid-state nuclear magnetic resonance spectroscopy to validate a structural model previously generated by combining in vitro and in silico data. Our results indicate that T4SScc is well folded in the cellular setting, revealing protein regions that had been elusive when studied in vitro.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Figure 1: Analysis of DNP-based two and three-dimensional ssNMR data sets of tailored-labeled cell-embedded T4SScc.
Figure 2: Summary of residue-specific ssNMR probes and their location in reference to the cellular envelope including the T4SScc electron microscopy map with the outer membrane complex fitted inside (PDB 3JQO).

Accession codes


Protein Data Bank


  1. Huang, B., Babcock, H. & Zhuang, X. Cell 143, 1047–1058 (2010).

    Article  CAS  Google Scholar 

  2. Leis, A., Rockel, B., Andrees, L. & Baumeister, W. Trends Biochem. Sci. 34, 60–70 (2009).

    Article  CAS  Google Scholar 

  3. Serber, Z., Corsini, L., Durst, F. & Dötsch, V. Methods Enzymol. 394, 17–41 (2005).

    Article  CAS  Google Scholar 

  4. Banci, L., Barbieri, L., Luchinat, E. & Secci, E. Chem. Biol. 20, 747–752 (2013).

    Article  CAS  Google Scholar 

  5. Etzkorn, M. et al. Angew. Chem. Int. Edn Engl. 46, 459–462 (2007).

    Article  CAS  Google Scholar 

  6. Fu, R. et al. J. Am. Chem. Soc. 133, 12370–12373 (2011).

    Article  CAS  Google Scholar 

  7. Jacso, T. et al. Angew. Chem. 124, 447–450 (2012).

    Article  Google Scholar 

  8. Miao, Y. et al. Angew. Chem. Int. Edn Engl. 51, 8383–8386 (2012).

    Article  CAS  Google Scholar 

  9. Renault, M. et al. Proc. Natl. Acad. Sci. USA 109, 4863–4868 (2012).

    Article  CAS  Google Scholar 

  10. Renault, M. et al. Angew. Chem. Int. Edn Engl. 51, 2998–3001 (2012).

    Article  CAS  Google Scholar 

  11. Ni, Q.Z. et al. Acc. Chem. Res. 46, 1933–1941 (2013).

    Article  CAS  Google Scholar 

  12. Fronzes, R. et al. Science 323, 266–268 (2009).

    Article  CAS  Google Scholar 

  13. Low, H.H. et al. Nature 508, 550–553 (2014).

    Article  CAS  Google Scholar 

  14. Rivera-Calzada, A. et al. EMBO J. 32, 1195–1204 (2013).

    Article  CAS  Google Scholar 

  15. Chandran, V. et al. Nature 462, 1011–1015 (2009).

    Article  CAS  Google Scholar 

  16. Trokter, M., Felisberto-rodrigues, C., Christie, P.J. & Waksman, G. Curr. Opin. Struct. Biol. 27, 16–23 (2014).

    Article  CAS  Google Scholar 

  17. Jakubowski, S.J. et al. Mol. Microbiol. 71, 779–794 (2009).

    Article  CAS  Google Scholar 

  18. Gradmann, S. et al. J. Biomol. NMR 54, 377–387 (2012).

    Article  CAS  Google Scholar 

  19. Shen, Y., Delaglio, F., Cornilescu, G. & Bax, A. J. Biomol. NMR 44, 213–223 (2009).

    Article  CAS  Google Scholar 

  20. Sauvée, C. et al. Angew. Chem. Int. Edn. Engl. 52, 10858–10861 (2013).

    Article  Google Scholar 

  21. Seidel, K., Etzkorn, M., Schneider, R., Ader, C. & Baldus, M. Solid State Nucl. Magn. Reson. 35, 235–242 (2009).

    Article  CAS  Google Scholar 

  22. Waksman, G. & Orlova, E.V. Curr. Opin. Microbiol. 17, 24–31 (2014).

    Article  CAS  Google Scholar 

  23. Llosa, M., Schröder, G. & Dehio, C. Trends Microbiol. 20, 355–359 (2012).

    Article  CAS  Google Scholar 

  24. Baldus, M., Petkova, A.T., Herzfeld, J. & Griffin, R.G. Mol. Phys. 95, 1197–1207 (1998).

    Article  CAS  Google Scholar 

  25. Weingarth, M., Demco, D.E., Bodenhausen, G. & Tekely, P. Chem. Phys. Lett. 469, 342–348 (2009).

    Article  CAS  Google Scholar 

  26. Bloembergen, N. Physica 15, 386–426 (1949).

    Article  CAS  Google Scholar 

  27. Fung, B.M., Khitrin, A.K. & Ermolaev, K. J. Magn. Reson. 142, 97–101 (2000).

    Article  CAS  Google Scholar 

  28. Cole, C., Barber, J.D. & Barton, G.J. Nucleic Acids Res. 36, W197–W201 (2008).

    Article  CAS  Google Scholar 

  29. Buchan, D.W., Minneci, F., Nugent, T.C.O., Bryson, K. & Jones, D.T. Nucleic Acids Res. 41, W349–W357 (2013).

    Article  Google Scholar 

  30. Shu, W., Liu, J., Ji, H. & Lu, M. J. Mol. Biol. 299, 1101–1112 (2000).

    Article  CAS  Google Scholar 

  31. Neal, S., Nip, A.M., Zhang, H. & Wishart, D.S. J. Biomol. NMR 26, 215–240 (2003).

    Article  CAS  Google Scholar 

  32. Pettersen, E.F. et al. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

Download references


We thank E. Koers and J. van der Zwan for helpful discussions and technical support. We are indebted to P. Tordo and his group for providing AMUPol. This work was funded in part by the Netherlands Organization for Scientific Research (NWO, grants 700.26.121 and 700.10.443 to M.B.)

Author information

Authors and Affiliations



G.E.F. and M.B. designed the research. M.K., M.D., A.C. and S.N. produced samples, and M.K. and D.M. performed ssNMR experiments. M.K., G.C.P.v.Z. and A.M.J.J.B. docked atomic models into the electron microscopy density map. R.F. provided clones and conducted electron microscopy studies. G.W. and M.B. wrote the paper and all authors edited it.

Corresponding authors

Correspondence to Gert E Folkers or Marc Baldus.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Cellular ssNMR method and the structure of T4SScc.

a) Scheme to use cellular ssNMR as a validation tool of structural models obtained by in-vitro and in-silico techniques in their native environment. b) Schematic view of T4SScc (VirB7, top, VirB9, middle, VirB10, bottom) where red and blue symbols represent helical and extended conformations of the crystallized part (PDB=3JQO). Grey regions and black crosses reflect consensus and non-consensus regions of different prediction servers, respectively.

Supplementary Figure 2 Sample preparation and characterization using EM.

a) Lane 1: 10% Tris/Glycine SDS-PAGE of cell envelope of BL21 WT expressing T4SScc. Lanes 2 and 3: 17.5% Tris/Tricine SDS-PAGE of cell envelope of BL21dm expressing T4SScc (lane 2) and without expressing T4SScc (lane 3). M refers to the molecular weight marker. b) Electron Microscopy data of T4SScc after purification

Supplementary Figure 3 Probing selective labeling for GSLV-T4SScc and TV-T4SScc in BL21dm cell envelopes by 2D ssNMR.

Two-dimensional ssNMR 13C-13C spin diffusion data on BL21dm cell envelopes a) expressing GSLV-T4SScc recorded at 700 MHz and b) expressing TV-T4SScc recorded at 800 MHz DNP. In a) intra-residue correlations of Gly, Leu, Ser and Val residues are indicated along with cross peaks consistent with labeled lipids signals. In b), black crosses represent FANDAS predictions for TV-T4SScc

Supplementary Figure 4 Prediction of the secondary structure elements of the inner layer complex of T4SScc.

Secondary structure prediction of the N-termini of VirB9 and VirB10 using different servers (JPred3 and PSIPRED).

Supplementary Figure 5 SDS-PAGE comparison of cell envelopes without and with expressing PagL and T4SScc.

Comparison of SDS-Page results of cellular envelope preparations without (- IPTG) and with (+ IPTG) PagL (a, Ref.) expression. b) Cell envelope preparation without (- tetracycline) and with (+ tetracycline) expression of T4SScc. We note that B7 only comprises 48 residues and it thus difficult to detect by Polyacrylamide gel electrophoresis (see also ref.). See also Supplementary Fig. 2.

Supplementary Figure 6 Comparison of 2D ssNMR data on uniformly [13C,15N] labeled BL21dm cell envelopes without and with T4SScc expression.

2D ssNMR 15N-13C (NCa) correlation data obtained on cell envelopes prepared from fully [13C,15N] labeled BL21dm cells without (a) and (b) with expression of T4SScc. The dashed red box represents the spectral region typical for Glycine correlations. Symbols reflect spectroscopic predictions on the basis of the Lpp crystal structure (a, red, see also ref.) and the T4SScc model (b, blue) discussed in the main text.

Supplementary Figure 7 SsNMR on fully labeled cell envelope of BL21dm expressing T4SScc.

2D carbon-carbon ssNMR correlation experiment of U-labeled T4SScc with FANDAS predictions for T4SScc (red), LPS (cyan), PE (blue) and PG (brown). We note that Lpp signals would superimpose with T4SSc signals.

Supplementary Figure 8 Amino-acid sequence of Braun’s lipoprotein (Lpp).

Note that for the case of (13C,15N-Thr, 15N-Val) labeling we expect no contributions of Lpp to the sequential correlations studied in Fig. 1a,c and d, main text). In the case of 13C,15N (Gly, Ser, Leu, Val) labeling (Fig. 1b and e), only 4 out of 55 sequential correlations stem from Lpp. These expected correlations would resonate in spectral regions different from the amino-acid stretches examined in Fig. 1e in the main text.

Supplementary Figure 9 Experimental DNP enhancement factors at 400 MHz and 800 MHz.

Signal enhancements using DNP at 800 MHz (a, c) and 400 MHz (b, d) of the GSLV labeled (a and b) and TV labeled (c and d) BL21dm cell envelope expressing T4SScc after addition of 20 mM AMUpol. In a), a 1D 13C CP spectrum was measured at 800 MHz with microwave irradiation off (blue) and on (red). An enhancement factor of 15 was obtained. In b) a 1D 13C CP spectrum of the same sample was measured at a 400 MHz instrument with microwave irradiation off (blue) and microwave irradiation on (red). Here an enhancement factor of 57 was obtained. c) and d) represent equivalent experiments for the TV labeled sample where an enhancement factor of 15 was obtained at 800 MHz DNP and 65 at 400 MHz DNP conditions.

Supplementary Figure 10 DNP spectrum corroborates the presence of a folded T4SScc in the cell envelope.

Example of an ω23 plane of a 3D NCACX experiment at 400 MHz DNP conditions on TV-T4SScc in BL21dm cell envelopes revealing intra-residue Threonine correlations that exhibit secondary Ca/Cb chemical shifts typical for β-sheets (blue) and α-helices (red).

Supplementary Figure 11 Comparison between 400 MHz and 800 MHz DNP spectra.

Comparison of 2D NCOCX data on selectively [13C,15N] GSLV-T4SScc at 800 MHz (red) and 400 MHz (blue) DNP conditions. In the latter case, lipid correlations are folded in due to a particular setting of the spectral window in the t1 evolution dimension.

Supplementary Figure 12 SsNMR experiments at higher temperatures suggest that T4SScc is largely rigid in the bacterial cell envelope.

a) 1D NCA experiment on BL21dm cell envelope expressing GSLV-T4SScc recorded at -5° C (blue spectrum) and 10° C (red spectrum). No significant change in the spectrum is observed between the two temperatures. This indicates that, at high temperatures, T4SScc is still largely rigid. Taking into consideration that about 25% of the signal in this sample is originating from the N-terminal part of B10, this implies a rigid N-terminal of B10 at this temperature. b) 1D 90° pulse on 15N performed on the same sample mentioned in a) at 10° C. A prominent sharp lipid peak is visible in the spectrum (~36 ppm), consistent with mobile lipids, and a small broad protein signal is seen at ~120 ppm. This observation further supports the notion of a rigid T4SScc in the cell envelope at high temperatures.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 and Supplementary Tables 1–8 (PDF 1340 kb)

Supplementary Data

Atomic models of the T4SS core complex in the EM density and of the N-terminal transmembrane helix of B10. The atomic model of the outer membrane complex of T4SScc (PDB 3JQO) was fitted into the EM density (EMD-2232) as a rigid body. The predicted transmembrane helix in the N-terminal part of VirB10 was modeled by imposing a helical conformation on the amino acid sequence. For the non-crystalline N-terminal part of VirB9, the already docked atomic model (PDB 2YPW)14 in the EM density (EMD = 2,232) was used. (ZIP 204 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kaplan, M., Cukkemane, A., van Zundert, G. et al. Probing a cell-embedded megadalton protein complex by DNP-supported solid-state NMR. Nat Methods 12, 649–652 (2015).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing