Skip to main content
SpringerLink
Log in

A protocol for the refinement of NMR structures using simultaneously pseudocontact shift restraints from multiple lanthanide ions

  • Article
  • Published:
Journal of Biomolecular NMR Aims and scope Submit manuscript

Abstract

The binding of paramagnetic metal ions to proteins produces a number of different effects on the NMR spectra of the system. In particular, when the magnetic susceptibility of the metal ion is anisotropic, pseudocontact shifts (PCSs) arise and can be easily measured. They constitute very useful restraints for the solution structure determination of metal-binding proteins. In this context, there has been great interest in the use of lanthanide(III) ions to induce PCSs in diamagnetic proteins, e.g. through the replacement native calcium(II) ions. By preparing multiple samples in each of which a different ion of the lanthanide series is introduced, it is possible to obtain multiple independent PCS datasets that can be used synergistically to generate protein structure ensembles (typically called bundles). For typical NMR-based determination of protein structure, it is necessary to perform an energetic refinement of such initial bundles to obtain final structures whose geometric quality is suitable for deposition in the PDB. This can be conveniently done by using restrained molecular dynamics simulations (rMD) in explicit solvent. However, there are no available protocols for rMD using multiple PCS datasets as part of the restraints. In this work, we extended the PCS module of the AMBER MD package to handle multiple datasets and tuned a previously developed protocol for NMR structure refinement to achieve consistent convergence with PCS restraints. Test calculations with real experimental data show that this new implementation delivers the expected improvement of protein geometry, resulting in final structures that are of suitable quality for deposition. Furthermore, we observe that also initial structures generated only with traditional restraints can be successfully refined using traditional and PCS restraints simultaneously.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Allegrozzi M, Bertini I, Janik MBL, Lee Y-M, Liu G, Luchinat C (2000) Lanthanide induced pseudocontact shifts for solution structure refinements of macromolecules in shells up to 40 A from the metal ion. J Am Chem Soc 122:4154–4161

    Article  Google Scholar 

  • Balayssac S, Bertini I, Bhaumik A, Lelli M, Luchinat C (2008) Paramagnetic shifts in solid-state NMR of proteins to elicit strucutral information. Proc Natl Acad Sci USA 105:17284–17289

    Article  ADS  Google Scholar 

  • Banci L, Bertini I, Bren KL, Cremonini MA, Gray HB, Luchinat C, Turano P (1996) The use of pseudocontact shifts to refine solution structures of paramagnetic metalloproteins: Met80Ala cyano-cytochrome c as an example. J Biol Inorg Chem 1:117–126

    Article  Google Scholar 

  • Banci L, Bertini I, Gori Savellini G, Romagnoli A, Turano P, Cremonini MA, Luchinat C, Gray HB (1997) Pseudocontact shifts as constraints for energy minimization and molecular dynamic calculations on solution structures of paramagnetic metalloproteins. Proteins Struct Funct Genet 29:68–76

    Article  Google Scholar 

  • Banci L, Bertini I, Cremonini MA, Gori Savellini G, Luchinat C, Wüthrich K, Güntert P (1998a) PSEUDODYANA for NMR structure calculation of paramagnetic metalloproteins using torsion angle molecular dynamics. J Biomol NMR 12:553–557

    Article  Google Scholar 

  • Banci L, Bertini I, Huber JG, Luchinat C, Rosato A (1998b) Partial orientation of oxidized and reduced cytochrome b5 at high magnetic fields: magnetic susceptibility anisotropy contributions and consequences for protein solution structure determination. J Am Chem Soc 120:12903–12909

    Article  Google Scholar 

  • Banci L, Bertini I, Cavallaro G, Giachetti A, Luchinat C, Parigi G (2004) Paramagnetism-based restraints for Xplor-NIH. J Biomol NMR 28:249–261

    Article  Google Scholar 

  • Barthelmes K, Reynolds AM, Peisach E, Jonker HRA, DeNunzio NJ, Allen KN, Imperiali B, Schwalbe H (2011) Engineering encodable lanthanide-binding tags into loop regions of proteins. J Am Chem Soc 133:808–819

    Article  Google Scholar 

  • Bertini I, Janik MBL, Lee Y-M, Luchinat C, Rosato A (2001a) Magnetic susceptibility tensor anisotropies for a lanthanide ion series in a fixed protein matrix. J Am Chem Soc 123:4181–4188

    Article  Google Scholar 

  • Bertini I, Janik MBL, Liu G, Luchinat C, Rosato A (2001b) Solution structure calculations through self-orientation in a magnetic field of cerium (III) substituted calcium-binding protein. J Magn Reson 148:23–30

    Article  ADS  Google Scholar 

  • Bertini I, Gelis I, Katsaros N, Luchinat C, Provenzani A (2003) Tuning the affinity for lanthanides of calcium binding proteins. Biochemistry 42:8011–8021

    Article  Google Scholar 

  • Bertini I, Luchinat C, Parigi G, Pierattelli R (2008) Perspectives in NMR of paramagnetic proteins. Dalton Trans 2008:3782–3790

    Article  Google Scholar 

  • Bertini I, Bhaumik A, De Paepe G, Griffin RG, Lelli M, Lewandowski JR, Luchinat C (2010) High-resolution solid-state NMR structure of a 17.6 kDa protein. J Am Chem Soc 132:1032–1040

    Article  Google Scholar 

  • Bertini I, Case DA, Ferella L, Giachetti A, Rosato A (2011) A grid-enable web portal for NMR structure refinement with AMBER. Bioinformatics 27:2384–2390

    Article  Google Scholar 

  • Bhattacharya A, Tejero R, Montelione GT (2007) Evaluating protein structures determined by structural genomics consortia. Proteins: Struct, Funct, Bioinf 66:778–795

    Article  Google Scholar 

  • Brewer KD, Bacaj T, Cavalli A, Camilloni C, Swarbrick JD, Liu J, Zhou A, Zhou P, Barlow N, Xu J, Seven AB, Prinslow EA, Voleti R, Haussinger D, Bonvin AM, Tomchick DR, Vendruscolo M, Graham B, Sudhof TC, Rizo J (2015) Dynamic binding mode of a Synaptotagmin-1-SNARE complex in solution. Nat Struct Mol Biol 22:555–564

    Article  Google Scholar 

  • Camilloni C, Vendruscolo M (2015) Using pseudocontact shifts and residual dipolar couplings as exact NMR restraints for the determination of protein structural ensembles. Biochemistry 54:7470–7476

    Article  Google Scholar 

  • Carlon A, Ravera E, Andralojc W, Parigi G, Murshudov GN, Luchinat C (2016) How to tackle protein structural data from solution and solid state: an integrated approach. Progress NMR Spectrosc 92–93:54–70

    Article  Google Scholar 

  • Cavalli A, Vendruscolo M (2015) Analysis of the performance of the CHESHIRE and YAPP methods at CASD-NMR round 3. J Biomol NMR 62:503–509

    Article  Google Scholar 

  • Cavalli A, Salvatella X, Dobson CM, Vendruscolo M (2007) Protein structure determination from NMR chemical shifts. Proc Natl Acad Sci USA 104:9615–9620

    Article  ADS  Google Scholar 

  • Cavanagh J, Fairbrother WJ, Palmer AG III, Rance M, Skelton NJ (2007) Protein NMR spectroscopy. Principles and practice. Academic Press, SanDiego

    Google Scholar 

  • Chen J, Im W, Brooks CL III (2004) Refinement of NMR structures using implicit solvent and advanced sampling techniques. J Am Chem Soc 126:16038–16047

    Article  Google Scholar 

  • Chen WN, Loscha KV, Nitsche C, Graham B, Otting G (2014) The dengue virus NS2B-NS3 protease retains the closed conformation in the complex with BPTI. FEBS Lett 588:2206–2211

    Article  Google Scholar 

  • Doreleijers JF, Sousa da Silva AW, Krieger E, Nabuurs SB, Spronk CA, Stevens TJ, Vranken WF, Vriend G, Vuister GW (2012) CING: an integrated residue-based structure validation program suite. J Biomol NMR 54:267–283

    Article  Google Scholar 

  • Feig M, Im W, Brooks CL III (2004) Implicit solvation based on generalized Born theory in different dielectric environments. J Chem Phys 120:903–911

    Article  ADS  Google Scholar 

  • Fossi M, Oschkinat H, Nilges M, Ball LJ (2005) Quantitative study of the effects of chemical shift tolerances and rates of SA cooling on structure calculation from automatically assigned NOE data. J Magn Reson 175:92–102

    Article  ADS  Google Scholar 

  • Gaponenko V, Sarma SP, Altieri AS, Horita DA, Li J, Byrd RA (2004) Improving the accuracy of NMR structures of large proteins using pseudocontact shifts as long/range restraints. J Biomol NMR 28:205–212

    Article  Google Scholar 

  • Gochin M, Roder H (1995) Use of pseudocontact shifts as a structural constraint for macromolecules in solution. Bull Magn Reson 17:1–4

    Google Scholar 

  • Hass MA, Ubbink M (2014) Structure determination of protein-protein complexes with long-range anisotropic paramagnetic NMR restraints. Curr Opin Struct Biol 24:45–53

    Article  Google Scholar 

  • Herrmann T, Güntert P, Wüthrich K (2002) 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

    Article  Google Scholar 

  • Huang YJ, Rosato A, Singh G, Montelione GT (2012) RPF—a quality assessment tool for protein NMR structures. Nucleic Acids Res 40:W542–W546

    Article  Google Scholar 

  • Hulsker R, Baranova MV, Bullerjahn GS, Ubbink M (2008) Dynamics in the transient complex of plastocyanin-cytochrome f from Prochlorothrix hollandica. J Am Chem Soc 130:1985–1991

    Article  Google Scholar 

  • Jao CC, Hedge BG, Chen J, Haworth IS, Langen R (2008) Structure of membrane-bound alpha-synuclein from site-directed spin labeling and computational refinement. Proc Natl Acad Sci USA 105:19666–19671

    Article  ADS  Google Scholar 

  • Jaroniec CP (2015) Structural studies of proteins by paramagnetic solid-state NMR spectroscopy. J Magn Reson 253:50–59

    Article  ADS  Google Scholar 

  • Kabsch W, Sander C (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22:2577–2637

    Article  Google Scholar 

  • Karplus M (1959) Contact electron-spin coupling of nuclear magnetic moments. J Chem Phys 30:11–15

    Article  ADS  Google Scholar 

  • Lange OF, Rossi P, Sgourakis NG, Song Y, Lee HW, Aramini JM, Ertekin A, Xiao R, Acton TB, Montelione GT, Baker D (2012) Determination of solution structures of proteins up to 40 kDa using CS-Rosetta with sparse NMR data from deuterated samples. Proc Natl Acad Sci USA 109:10873–10878

    Article  ADS  Google Scholar 

  • Li J, Pilla KB, Li Q, Zhang Z, Su X, Huber T, Yang J (2013) Magic angle spinning NMR structure determination of proteins from pseudocontact shifts. J Am Chem Soc 135:8294–8303

    Article  Google Scholar 

  • Linge JP, Nilges M (1999) Influence of non-bonded parameters on the quality of NMR structures: a new force field for NMR structure calculation. J Biomol NMR 13:51–59

    Article  Google Scholar 

  • Linge JP, Williams MA, Spronk CAEM, Bonvin AMJJ, Nilges M (2003) Refinement of protein structures in explicit solvent. Proteins: Struct, Funct, Bioinf 50:496–506

    Article  Google Scholar 

  • Mao B, Tejero R, Baker D, Montelione GT (2014) Protein NMR structures refined with Rosetta have higher accuracy relative to corresponding X-ray crystal structures. J Am Chem Soc 136:1893–1906

    Article  Google Scholar 

  • Mareuil F, Malliavin TE, Nilges M, Bardiaux B (2015) Improved reliability, accuracy and quality in automated NMR structure calculation with ARIA. J Biomol NMR 62:425–438

    Article  Google Scholar 

  • McConnell HM, Robertson RE (1958) Isotropic nuclear resonance shifts. J Chem Phys 29:1361–1365

    Article  ADS  Google Scholar 

  • Montelione GT, Nilges M, Bax A, Guntert P, Herrmann T, Richardson JS, Schwieters CD, Vranken WF, Vuister GW, Wishart DS, Berman HM, Kleywegt GJ, Markley JL (2013) Recommendations of the wwPDB NMR validation task force. Structure 21:1563–1570

    Article  Google Scholar 

  • Muntener T, Haussinger D, Selenko P, Theillet FX (2016) In-cell protein structures from 2D NMR Experiments. J Phys Chem Lett 7:2821–2825

    Article  Google Scholar 

  • Nabuurs SB, Nederveen AJ, Vranken W, Doreleijers JF, Bonvin AM, Vuister GW, Vriend G, Spronk CA (2004) DRESS: a database of REfined solution NMR structures. Proteins: Struct, Funct, Bioinf 55:483–486

    Article  Google Scholar 

  • Nabuurs SB, Spronk CA, Vuister GW, Vriend G (2006) Traditional biomolecular structure determination by NMR spectroscopy allows for major errors. PLoS Comput Biol 2:e9

    Article  ADS  Google Scholar 

  • Nederveen AJ, Doreleijers JF, Vranken W, Miller Z, Spronk CA, Nabuurs SB, Güntert P, Livny M, Markley JL, Nilges M, Ulrich EL, Kaptein R, Bonvin AM (2005) RECOORD: a recalculated coordinate database of 500 + proteins from the PDB using restraints from the BioMagResBank. Proteins: Struct, Funct, Bioinf 59:662–672

    Article  Google Scholar 

  • Pan BB, Yang F, Ye Y, Wu Q, Li C, Huber T, Su XC (2016) 3D structure determination of a protein in living cells using paramagnetic NMR spectroscopy. Chem Commun (Camb) 52:10237–10240

    Article  Google Scholar 

  • Ragan TJ, Fogh RH, Tejero R, Vranken W, Montelione GT, Rosato A, Vuister GW (2015) Analysis of the structural quality of the CASD-NMR 2013 entries. J Biomol NMR 62:527–540

    Article  Google Scholar 

  • Rinaldelli M, Ravera E, Calderone V, Parigi G, Murshudov GN, Luchinat C (2014) Simultaneous use of solution NMR and X-ray data REFMAC5 for joint refinement/detection of structural differences. Acta Cryst D D70:958–967

    Article  Google Scholar 

  • Rinaldelli M, Carlon A, Ravera E, Parigi G, Luchinat C (2015) FANTEN: a new web-based interface for the analysis of magnetic anisotropy-induced NMR data. J Biomol NMR 61:21–34

    Article  Google Scholar 

  • Rodriguez-Castañeda F, Haberz P, Leonov A, Griesinger C (2006) Paramagnetic tagging of diamagnetic proteins for solution NMR. Magn Reson Chem 44:S10–S16

    Article  Google Scholar 

  • Rosato A, Aramini JM, Arrowsmith C, Bagaria A, Baker D, Cavalli A, Doreleijers JF, Eletsky A, Giachetti A, Guerry P, Gutmanas A, Guntert P, He Y, Herrmann T, Huang YJ, Jaravine V, Jonker HR, Kennedy MA, Lange OF, Liu G, Malliavin TE, Mani R, Mao B, Montelione GT, Nilges M, Rossi P, van der Schot G, Schwalbe H, Szyperski TA, Vendruscolo M, Vernon R, Vranken WF, de Vries S, Vuister GW, Wu B, Yang Y, Bonvin AMJJ (2012) Blind testing of routine, fully automated determination of protein structures from NMR data. Structure 20:227–236

    Article  Google Scholar 

  • Rosato A, Tejero R, Montelione GT (2013) Quality assessment of protein NMR structures. Curr Opin Struct Biol 23:715–724

    Article  Google Scholar 

  • Ryu H, Lim G, Sung BH, Lee J (2016) NMRe: a web server for NMR protein structure refinement with high-quality structure validation scores. Bioinformatics 32:611–613

    Article  Google Scholar 

  • Saccenti E, Rosato A (2008) The war of tools: how can NMR spectroscopists detect errors in their structures? J Biomol NMR 40:251–261

    Article  Google Scholar 

  • Schmitz C, Vernon R, Otting G, Baker D, Huber T (2012) Protein structure determination from pseudocontact shifts using ROSETTA. J Mol Biol 416:668–677

    Article  Google Scholar 

  • Serrano P, Dutta SK, Proudfoot A, Mohanty B, Susac L, Martin B, Geralt M, Jaroszewski L, Godzik A, Elsliger M, Wilson IA, Wuthrich K (2016) NMR in structural genomics to increase structural coverage of the protein universe. FEBS J. doi:10.1111/febs.13751

    Google Scholar 

  • Shen Y, Lange O, Delaglio F, Rossi P, Aramini JM, Liu G, Eletsky A, Wu Y, Singarapu KK, Lemak A, Ignatchenko A, Arrowsmith CH, Szyperski T, Montelione GT, Baker D, Bax A (2008) Consistent blind protein structure generation from NMR chemical shift data. Proc Natl Acad Sci USA 105:4685–4690

    Article  ADS  Google Scholar 

  • Shen Y, Delaglio F, Cornilescu G, Bax A (2009) TALOS + : a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J Biomol NMR 44:213–223

    Article  Google Scholar 

  • Su XC, Otting G (2010) Paramagnetic labelling of proteins and oligonucleotides for NMR. J Biomol NMR 46:101–112

    Article  Google Scholar 

  • Synder DA, Chen Y, Denissova NG, Acton T, Aramini JM, Ciano M, Karlin R, Liu J, Manor P, Rajan PA, Rossi P, Swapna GV, Xiao R, Rost B, Hunt J, Montelione GT (2005) Comparisons of NMR spectral quality and success in crystallization demonstrate that NMR and X-ray crystallography are complementary methods for small protein structure determination. J Am Chem Soc 127:16505–16511

    Article  Google Scholar 

  • Tang Y, Huang YJ, Hopf TA, Sander C, Marks DS, Montelione GT (2015) Protein structure determination by combining sparse NMR data with evolutionary couplings. Nat Methods 12:751–754

    Article  Google Scholar 

  • Tjandra N, Bax A (1997) Direct measurement of distances and angles in biomolecules by NMR in a diluite liquid crystalline medium. Science 278:1111–1114

    Article  ADS  Google Scholar 

  • van der Schot G, Bonvin AM (2015) Performance of the WeNMR CS-Rosetta3 web server in CASD-NMR. J Biomol NMR 62:497–502

    Article  Google Scholar 

  • Vernon R, Shen Y, Baker D, Lange OF (2013) Improved chemical shift based fragment selection for CS-Rosetta using Rosetta3 fragment picker. J Biomol NMR 57:117–127

    Article  Google Scholar 

  • Vuister GW, Fogh RH, Hendrickx PM, Doreleijers JF, Gutmanas A (2014) An overview of tools for the validation of protein NMR structures. J Biomol NMR 58:259–285

    Article  Google Scholar 

  • Wassenaar TA, van Dijk M, Loureiro-Ferreira N, van der Schot G, de Vries SJ, Schmitz C, van der Zwan J, Boelens R, Giachetti A, Ferella L, Rosato A, Bertini I, Herrmann T, Jonker HRA, Bagaria A, Jaravine V, Guntert P, Schwalbe H, Vranken WF, Doreleijers JF, Vriend G, Vuister GW, Franke D, Kikhney A, Svergun DI, Fogh RH, Ionides J, Laue ED, Spronk C, Jurksa S, Verlato M, Badoer S, Dal Pra S, Mazzucato M, Frizziero E, Bonvin AMJJ (2012) WeNMR: structural biology on the grid. J Grid Comput 10:743–767

    Article  Google Scholar 

  • Wüthrich K (1986) NMR of proteins and nucleic acids. Wiley, New York

    Google Scholar 

  • Xia B, Tsui V, Case DA, Dyson HJ, Wright PE (2002) Comparison of protein solution structures refined by molecular dynamics simulation in vacuum, with a generalized Born model, and with explicit water. J Biomol NMR 22:317–331

    Article  Google Scholar 

  • Yagi H, Pilla KB, Maleckis A, Graham B, Huber T, Otting G (2013) Three-dimensional protein fold determination from backbone amide pseudocontact shifts generated by lanthanide tags at multiple sites. Structure 21:883–890

    Article  Google Scholar 

  • Yee AA, Savchenko A, Ignatchenko A, Lukin J, Xu X, Skarina T, Evdokimova E, Liu CS, Semesi A, Guido V, Edwards AM, Arrowsmith CH (2005) NMR and X-ray crystallography, complementary tools in structural proteomics of small proteins. J Am Chem Soc 127:16512–16517

    Article  Google Scholar 

Download references

Acknowledgments

This work has been supported by the European Commission through contracts EGI-Engage No. 654142 and WeNMR No. 261572. The FP7 WeNMR and H2020 West-Life (no. 675858) European e-Infrastructure projects are acknowledged for the use of their web portals, which make use of the EGI infrastructure and DIRAC4EGI service with the dedicated support of CESNET-MetaCloud, INFN-PADOVA, NCG-INGRID-PT, RAL-LCG2, TW-NCHC, SURFsara and NIKHEF, and the additional support of the national GRID Initiatives of Belgium, France, Italy, Germany, the Netherlands, Poland, Portugal, Spain, UK, South Africa, Malaysia, Taiwan and the US Open Science Grid.

Author information

Authors and Affiliations

  1. Magnetic Resonance Center (CERM), University of Florence, Via Luigi Sacconi 6, 50019, Sesto Fiorentino, Italy

    Davide Sala, Andrea Giachetti, Claudio Luchinat & Antonio Rosato

  2. Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019, Sesto Fiorentino, Italy

    Claudio Luchinat & Antonio Rosato

Authors
  1. Davide Sala
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrea Giachetti
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Claudio Luchinat
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Antonio Rosato
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Claudio Luchinat or Antonio Rosato.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 5030 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sala, D., Giachetti, A., Luchinat, C. et al. A protocol for the refinement of NMR structures using simultaneously pseudocontact shift restraints from multiple lanthanide ions. J Biomol NMR 66, 175–185 (2016). https://doi.org/10.1007/s10858-016-0065-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-016-0065-6

Keywords

Search

Navigation