b) NMR: prediction of molecular alignment from structure

More documents

Recommendations

Info

© 2008 Nature Publishing Group http: / / www. nature. com/ natureprotocols PROTOCOL TABLE 5 | Troubleshooting table (continued). Step Problem Possible reason Solution Number of RDCs in RDC input table does not match number of RDCs selected by PALES PALES reports ‘REMARK 0 couplings selected (inclusive max)’ on the command line 5 The correlation between experimental and back-calculated RDCs is unexpectedly low The RDC quality factor reported by PALES differs from that calculated by other programs ‘PALES –pdb pdb1ubqH.ent –inD dObs.tab’ is executed, but PALES appears to perform alignment prediction instead of best-fitting RDCs 5, 10, 17 The output file (e.g., ‘dCalc.Svd.tab’) is empty except for a few remark lines The dipolar interaction values (column ‘DD’ in output file) all have the same value, although the internuclear distances in the PDB file are different 7 In ‘rot.pdb’, only one molecule is present, although the output file (e.g., ‘dCalc.Svd.tab’) contains four sets of Euler angles 10, 17 No RDCs but the alignment tensor parameters are reported in the output file (e.g., ‘dCalc.Steric.tab’) 11, 20 The number of atoms selected by PALES (e.g., ‘REMARK 113 atoms selected for simulation’) is almost invariant to the removal of certain parts of the molecule 14 There is no charge assigned to the N-terminal amino group of the protein in the charge file 14, 15 PALES reports ‘WARNING: There is no residue 45 or the proper atom in the given PDB file!’. 16 PALES reports ‘Warning: Potentials between neighboring cells overlapping, cutoff 6.000000e- 06! ---4 Magnitude of alignment tensor wrong!’ 688 | VOL.3 NO.4 | 2008 | NATURE PROTOCOLS Atom names in RDC input file are not identical to those in the PDB file (e.g., amide protons are named ‘H’ and not ‘HN’) Atom names or residue numbers in RDC input file are not identical to those in the PDB file Wrong structure selected. Alternatively, residue numbering in RDC input table differs from that in PDB file Different definitions of the RDC quality factor Adjust atom names and residue numbers in RDC input table Adjust atom names in RDC input table Select correct structure. Correct for offset in residue numbering using the ‘-s1’ and‘-a1’ PALES flags Try out two other methods for calculation of the RDC quality factor by specifying the ‘-qRms’ orthe‘-qStd’ flag when running PALES The ‘-bestFit’ flag was not included Include the ‘-bestFit’ flag when running PALES PALES selected zero RDCs from the input table PALES automatically adjusted the distances between backbone heavy atoms and their hydrogen atoms PALES randomly selects one of the four possible orientations Adjust atom names in RDC input table Include the ‘-nofixedDI’ flag when running PALES Include the ‘-rotID’ flag and specify the rotation number (from 0 to 3) you would like to select (e.g., ‘-rotID 2’) No RDC input table was specified Supply an RDC input table (Step 2). This is not used for the simulation itself, but tells PALES which RDCs you are interested in PALES selected only surface accessible atoms for the simulation Residue numbers in the PDB file do not start with 1 A residue number or atom name has been specified in the charge file that is not present in the PDB file Concentration of alignment medium (specified by the ‘-wv’ flag) is too high for the used ionic strength Tell PALES to use all atoms by including the ‘-nosurf’ flag Manually assign a positive charge to the nitrogen atom of the first residue Modify charge file or PDB file Reduce the concentration of the alignment medium or the ionic strength at which the simulation is performed
© 2008 Nature Publishing Group http: / / www. nature. com/ natureprotocols ANTICIPATED RESULTS An order matrix describes the preferred orientation of molecules (proteins, nucleic acids, oligosaccharides, small molecules) that have been dissolved in media (dilute liquid crystalline phases, anisotropic gels) that preferentially interact with the solute through steric and electrostatic interactions. When the user defines which internuclear vectors he or she is interested in (by supplying an RDC input table), PALES also calculates RDCs from the predicted alignment matrices. Additional information about a predicted alignment tensor and RDCs can be found in the output files (Table 4 and Supplementary Data). This includes the PALES simulation parameters, the irreducible representation of the order matrix, Sauson–Flamsteed coordinates to visualize tensor orientations, the tensor eigensystem and its corresponding Euler angles, and various quantities for quality assessment of calculated RDCs: root-mean-square deviation, Pearson’s linear correlation coefficient and RDC quality factor (Fig. 3). Note: Supplementary information is available via the HTML version of this article. ACKNOWLEDGMENTS I am grateful to Ad Bax for his guidance during my postdoctoral stay in his lab and his continuous support to develop PALES. Many thanks also to Frank Delaglio for useful discussions and access to source code handling input/output of dipolar couplings and PDB files, as well as best-fit of dipolar couplings to PDB files. This work was supported by the Max Planck Society and the DFG through grants ZW71/1-1 to 3-1. Published online at http://www.natureprotocols.com Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions 1. Wuthrich, K. NMR studies of structure and function of biological macromolecules (Nobel Lecture). Angew. Chem. Int. Ed. 42, 3340–3363 (2003). 2. Tjandra, N. & Bax, A. Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278, 1111–1114 (1997). 3. Tolman, J.R., Flanagan, J.M., Kennedy, M.A. & Prestegard, J.H. Nuclear magnetic dipole interactions in field-oriented proteins—information for structure determination in solution. Proc. Natl. Acad. Sci. USA 92, 9279–9283 (1995). 4. Prestegard, J.H., Al-Hashimi, H.M. & Tolman, J.R. NMR structures of biomolecules using field oriented media and residual dipolar couplings. Q. Rev. Biophys. 33, 371–424 (2000). 5. Bax, A., Kontaxis, G. & Tjandra, N. Nuclear magnetic resonance of biological macromolecules. Part B, 127–174 (2001). 6. Bothner-By, A.A. Magnetic field induced alignment of molecules. In Encyclopedia of Nuclear Magnetic Resonance. (eds. Grant, D.M. & Harris, R.K.) 2932–2938 (Wiley, Chichester, 1996). 7. Saupe, A. & Englert, G. Phys. Rev. Lett. 11, 462–464 (1963). 8. Tjandra, N., Omichinski, J.G., Gronenborn, A.M., Clore, G.M. & Bax, A. Use of dipolar H-1-N-15 and H-1-C-13 couplings in the structure determination of magnetically oriented macromolecules in solution. Nat. Struct. Biol. 4, 732–738 (1997). 9. Delaglio, F., Kontaxis, G. & Bax, A. Protein structure determination using molecular fragment replacement and NMR dipolar couplings. J. Am. Chem. Soc. 122, 2142–2143 (2000). 10. Cornilescu, G., Marquardt, J.L., Ottiger, M. & Bax, A. Validation of protein structure from anisotropic carbonyl chemical shifts in a dilute liquid crystalline phase. J. Am. Chem. Soc. 120, 6836–6837 (1998). 11. Fischer, M.W.F., Losonczi, J.A., Weaver, J.L. & Prestegard, J.H. Domain orientation and dynamics in multidomain proteins from residual dipolar couplings. Biochemistry 38, 9013–9022 (1999). 12. Meiler, J., Prompers, J.J., Peti, W., Griesinger, C. & Bruschweiler, R. Model-free approach to the dynamic interpretation of residual dipolar couplings in globular proteins. J. Am. Chem. Soc. 123, 6098–6107 (2001). RDC predicted [Hz] 60 40 20 0 –20 –40 –20 0 20 RDC experimental [Hz] PROTOCOL Figure 3 | Comparison between experimental one-bond 1 H- 15 NRDCsand values predicted from the 3D charge distribution and shape of the 76-residue protein ubiquitin (PDB code: 1D3Z; mean structure). Experimental values were measured in 5 mg ml 1 Pf1 bacteriophage, 50 mM NaCl, 10 mM NaH2PO4/ Na 2HPO 4,pH6.5. 13. Mohana-Borges, R., Goto, N.K., Kroon, G.J.A., Dyson, H.J. & Wright, P.E. Structural characterization of unfolded states of apomyoglobin using residual dipolar couplings. J. Mol. Biol. 340, 1131–1142 (2004). 14. Bertoncini, C.W. et al. Release of long-range tertiary interactions potentiates aggregation of natively unstructured alpha-synuclein. Proc. Natl. Acad. Sci. USA 102, 1430–1435 (2005). 15. Zweckstetter, M. & Bax, A. Prediction of sterically induced alignment in a dilute liquid crystalline phase: aid to protein structure determination by NMR. J. Am. Chem. Soc. 122, 3791–3792 (2000). 16. Ferrarini, A. Modeling of macromolecular alignment in nematic virus suspensions. Application to the prediction of NMR residual dipolar couplings. J. Phys. Chem. B 107, 7923–7931 (2003). 17. Zweckstetter, M., Hummer, G. & Bax, A. Prediction of charge-induced molecular alignment of biomolecules dissolved in dilute liquid crystalline phases. Biophys. J. 86, 3444–3460 (2004). 18. Zweckstetter, M. Prediction of charge-induced molecular alignment: residual dipolar couplings at pH 3 and alignment in surfactant liquid crystalline phases. Eur. Biophys. J. 35, 170–180 (2006). 19. Wu, B., Petersen, M., Girard, F., Tessari, M. & Wijmenga, S.S. Prediction of molecular alignment of nucleic acids in aligned media. J. Biomol. NMR 35, 103–115 (2006). 20. Almond, A. & Axelsen, J.B. Physical interpretation of residual dipolar couplings in neutral aligned media. J. Am. Chem. Soc. 124, 9986–9987 (2002). 21. Azurmendi, H.F. & Bush, C.A. Tracking alignment from the moment of inertia tensor (TRAMITE) of biomolecules in neutral dilute liquid crystal solutions. J. Am. Chem. Soc. 124, 2426–2427 (2002). 22. Fernandes, M.X., Bernado, P., Pons, M. & de la Torre, J.G. An analytical solution to the problem of the orientation of rigid particles by planar obstacles. Application to membrane systems and to the calculation of dipolar couplings in protein NMR spectroscopy. J. Am. Chem. Soc. 123, 12037–12047 (2001). 23. Bewley, C.A. Rapid validation of the overall structure of an internal domain-swapped mutant of the anti-HIV protein cyanovirin-N using residual dipolar couplings. J. Am. Chem. Soc. 123, 1014–1015 (2001). 24. Bewley, C.A. & Clore, G.M. Determination of the relative orientation of the two halves of the domain-swapped dimer of cyanovirin-N in solution using dipolar couplings and rigid body minimization. J. Am. Chem. Soc. 122, 6009–6016 (2000). 25. Valafar, H. & Prestegard, J.H. Rapid classification of a protein fold family using a statistical analysis of dipolar couplings. Bioinformatics 19, 1549–1555 (2003). 26. Warren, J.J. & Moore, P.B. Application of dipolar coupling data to the refinement of the solution structure of the Sarcin–Ricin loop RNA. J. Biomol. NMR 20, 311–323 (2001). 27. van Buuren, B.N.M. et al. NMR spectroscopic determination of the solution structure of a branched nucleic acid from residual dipolar couplings by using isotopically labeled nucleotides. Angew. Chem. Int. Ed. 43, 187–192 (2004). NATURE PROTOCOLS | VOL.3 NO.4 | 2008 | 689
Page 1 and 2: © 2008 Nature Publishing Group htt
Page 9: © 2008 Nature Publishing Group htt

b) NMR: prediction of molecular alignment from structure

Create successful ePaper yourself

Delete template?

Save as template?