Thank you Edward, Really appreciate the help and advice.
Best regards, Andras > On Apr 18, 2019, at 11:33 AM, Edward d'Auvergne <[email protected]> wrote: > > External Email - Use Caution > >> 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? > > As I mentioned to Thibault, book some more NMR time (~2 hours) and > rerun mini-versions of your experiments with MeOH or methylene glycol > standards. Then measure the temperature of the experiment. But if > you did minimally use fid interleaving for all experiments (single > scan interleaving is really only needed for the R2), then you will > have daily temperature fluctuations affecting your data in a > significant way. There is a publication on this effect by Wüthrich, I > think. > > >> 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. > > The local tm models are noisy - it is over-fitting - but the general > trend in the data should explain the global diffusion tensor selected. > Was the chi2 value high for the local tm models too? That would not > good and might indicate that you have bad data. > > >> 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. > > What about the chi2 values? Are these high? If not, that would again > be a sign of the individual experiments being ok, but that temperature > calibration and control might be an issue. > > >> 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. > > That is what I understood - the 15N-13C dipolar relaxation. If you > have fully labelled 15N and 13C samples, this is actually a > significant source of relaxation. Significant in a model-free > analysis would probably be a contribution of >5%. There are multiple > publications on this issue, but unfortunately I cannot remember them > off the top of my head. For example there was some work on this for > analysing DNA. I know this because there is an abandoned branch of > relax for handling multi-pole relaxation due to this exact effect in > nucleic acids. > > Anyway, you should first determine if your data is good or bad. You > could try the consistency testing analysis in relax for example. But > first carefully check your temperature calibration and control. And > also study the literature on 15N-13C dipolar relaxation in fully > labelled samples. And, as I mentioned to Thibault, no one gets > relaxation data measurements correct the first time ;) > > 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. _______________________________________________ nmr-relax-users mailing list [email protected] https://lists.sourceforge.net/lists/listinfo/nmr-relax-users
