Dear Edward, Thank you very much for your detailed reply. I'll try to reply point by point:
Temperature control: I did have a recent temperature calibration in place, but did not calibrate when setting up the experiment. I probably should have done that. I did use heat compensation blocks during the experiment, but did not do single scan or experiment interleaving. Is there a way to account for this in relax, or is re-recording of the data the only remedy? Single diffusion tensor estimate: I looked through the local tm model results and they appear to be all over the place, although the relaxation behavior of the protein predicts that they should be rather similar. chi2 values were generally in the double to three (hundreds) digit region. The model selection changes from residue to residue. I don't believe that this sample dimerizes, but have not done a dilution series yet. R1, R2, NOE: All fittings were done in Relax and heights and noise were measured following recommendations in the relax manual. I also compared fits to fitting with other methods. The errors and curve fits look ok. 13C Coupling: I think I did not formulate this question clearly. My concern was with the effect of dipolar couplings from 13C carbons on relaxation. There is no question that the scalar coupling must be taken care of at the experimental level and this is of course what I did. Upon looking into this further, I believe that the effect from the dipolar couplings should be negligible. With best regards, Andras On Apr 16, 2019, at 9:32 AM, Edward d'Auvergne <[email protected]<mailto:[email protected]>> wrote: External Email - Use Caution On Mon, 15 Apr 2019 at 19:18, Boeszoermenyi, Andras <[email protected]<mailto:[email protected]>> wrote: Dear Relax Community, I am trying to do model free analysis with relax 4.0.3 on a linux workstation in script mode. I have a high resolution (1.1 Angstrom) crystal structure of a 30 kDa protein and added protons to the pdb. For the model free I have T1, T2, HetNOE data sets at 600 MHz and 750 MHz (both spectrometers were Bruker) and all values make sense. The analysis of the data fits well, with small standard deviations for the vast majority of residues. I am using the "black-bocks" dauvergne-protocol and all calculations run perfectly fine. However, the Chi-squared test produces ridiculously high chi-squares ~17000 and then of course the wrong model is selected. Hi Andras, This could be due to a number of factors: - Missing or poor temperature calibration or control (see the "Temperature control and calibration" section of the "Relaxation curve-fitting" chapter of the manual, i.e. http://www.nmr-relax.com/manual/Temperature_control_and_calibration.html ). - Inadequacy of the single diffusion tensor estimate. Please compare the local tm model results to the final selected diffusion model. What is the total chi2 value for the local tm model? Could there be partial dimerisation, even if it is non-specific? - Incorrect errors in the R1, R2, and steady-state NOE values. Did you use relax to obtain this data? And how did you perform the error analysis? If the errors are too small, the final chi-squared value will be very high and the model-free models chosen will be wrong. See the "From spectra to peak intensities for the relaxation rates" section of the "Relaxation curve-fitting" chapter of the manual, i.e. http://www.nmr-relax.com/manual/From_spectra_to_peak_intensities_for_the_relaxation_rates.html . And, for example, the "NOE script mode - setting the errors" section of the "Calculating the NOE" chapter of the manual, i.e. http://www.nmr-relax.com/manual/NOE_script_mode_setting_the_errors.html . There are other factors as well. I suggest looking at the relaxation curves for the R1 and R2 very carefully, including error bars, to see if there is any issue with the base data. These curves are produced automatically by the relax auto-analyses and can be visualised in XMGrace. The steady-state NOE data and parameter plots should also be carefully checked. Did anyone make this experience, and would any one have a suggestion on how to fix it? See above. This has been seen many, many times before. The above 3 reasons are probably the most common ones - with temperature calibration and control being the number one culprit. You may be able to find the old threads on the relax users mailing list archive on this subject: https://sourceforge.net/p/nmr-relax/mailman/nmr-relax-users/ One more information. I ran the NMR experiments on a perdeuterated and 13C, 15N labeled sample. I did not find a way to correct for 13C couplings in the model free script. Might that have a role in this, and is there a way to correct for that? This will be an issue. I have never encountered literature on the subject of dealing with this on the data analysis level. I thought that this is usually dealt with by minimising the effect during the experiment. Do you have any references/papers that state otherwise? I would be interested in reading about it. I hope the above info helps. Regards, Edward The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail.
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