Dear Soumya. I know that meeting 'relax' can be quite overwhelming.
In our NMR lab, I have tried to make some small wiki-pages, to get our other Master/PhD/Postdocs up and running. The first problem to tackle, is to get the measure error of your spectrums. Have you recorded in dublicate/triplicate? or are you interested in using the spectrum noise as error estimator? http://wiki.nmr-relax.com/Spectrum_error_analysis As Edward suggest, you should investigate using SPARKY's extension for this: http://wiki.nmr-relax.com/RMSD Make for example a text which contain columns of "settings" for all your spectrums in a measurement (R1, R2, NOE). This file we can then loop over later, when making settings in relax. path_to_ft2_file spectrum_id spectrometer_frq relax_time rmsd_err Then you should be able to modify the script here: http://wiki.nmr-relax.com/Tutorial_for_R1/R2_Relaxation_curve-fitting_analysis_on_varian_recorded_as_fid_interleaved For a R1/R2 analysis. The output files "*.out" from these analysis you can the load in easily for the model-free analysis. For the NOE analysis, this i written in the manual. http://www.nmr-relax.com/manual/NOE_script_mode_sample_script.html Best Troels 2014-03-05 11:17 GMT+01:00 Edward d'Auvergne <[email protected]>: > Hi Soumya, > > I've had a close look at your relaxation data and model-free results > attached to https://gna.org/support/?3127, and have some comments > below: > > > > I've conducted model-free analysis on relax using relaxation data (T1, > T2 adn > > NOE) collected on 600 and 800 MHz instruments. > > Before I get to the other issues, I can see problems with the base > data. How did you calculate the T1, T2, and NOE? And more > importantly how did you calculate the errors? I would recommend you > try to calculate these again using relax, and see if you get the same > results. It is clear that your steady-state NOE errors are too small, > they are not even visible. If you have duplicated or triplicated > spectra, relax can use these to obtain an error estimate. > > Or you can perform the normal procedure of measuring the RMSD of the > baseplane noise. The 'rm' command in Sparky is the most powerful for > this. Do not use the estimates that spectral software give for the > entire spectrum, that is not accurate enough for a model-free analysis > (you must avoid peaks and the water signal, and it is unknown how > these blackbox estimates do that, or even if they do that at all). > You need to draw many boxes in empty parts of the spectrum, but close > to your peaks in the centre where it is noisier, and use an average > RMSD value from that. You can then input the RMSD into the NOE > analysis in relax and obtain the real NOE errors. > > > > I used the GUI with the default > > settings except for estimated rotational correlation time (for me it is > 5 ns). > > Note that model-free analysis consists of a complex iterative > protocol. Here is the protocol used in the GUI: > > http://www.nmr-relax.com/manual/Model_free_analysis_in_reverse.html > > You can read about it in the GUI by clicking on the 'About' button in > a model-free analysis tab. I published this protocol in the paper: > > - d'Auvergne, E. J. and Gooley, P. R. (2008). Optimisation of NMR > dynamic models II. A new methodology for the dual optimisation of the > model-free parameters and the Brownian rotational diffusion tensor. J. > Biomol. NMR, 40(2), 121-133. > (http://dx.doi.org/10.1007/s10858-007-9213-3). > > The key to this protocol is that the chicken or egg problem - which > comes first, the diffusion tensor or internal motion - is reversed > compared to how protocols were constructed in the past. This protocol > optimises the internal motion first and then the diffusion tensor. At > no point do you specify a diffusion tensor - the protocol will find > the correct one by itself. Correlation time estimates from other > biophysical techniques are always different to that found in the NMR > sample, due to concentration differences and microviscosity, so you > should not use these as starting points (which cannot be done in this > protocol anyway). > > > > My S2 order parameters are the inverse of what I expect. Disordered > regions > > appear to have values closer to one and regions I know are structured > have > > values that are closer to zero (see attached). The errors associated > with the > > "ordered" NHs are quite large whereas those for dynamic residues are > smaller. > > This appears to be the classic problem of inputting the data in the > wrong format - specifically when relaxation times and not relaxation > rates are input into the software. Rates must always be used. The > reason is that all of the fundamental NMR theory going all the way > back to the bible of NMR (Abragam) is based on rates. Spectral > density values add to give rates. Times are not the natural unit for > the physics of the system. If you use relax to recalculate all of the > R1, R2, and NOE data, then this problem should disappear. > > > > Here's what I observed. The calculation ran without any errors. It took > about > > three days to run with a single processor. Is that normal or is there > > something wrong here? > > 3 days is normal. It can take anywhere between a few hours on a > multiprocessor machine to 1-2 weeks. It depends on the molecular > system being used, the real diffusion tensor, how complicated the > internal dynamics are, and if the system is not perfectly described by > the classic spherical, spheroidal or ellipsoidal diffusion tensors. > It takes a long time because this is a full iterative protocol being > executed. And relax has far higher accuracy than any of the other > model-free software (see http://dx.doi.org/10.1007/s10858-007-9214-2). > If you have a multi-core or hyperthreaded system, you should try to > run Gary Thompson's multiprocessor for relax. If you have 8 cores, > you can run the relax model-free calculations 7 times faster (one of > the 8 is used for the master processor, > seehttp://www.nmr-relax.com/manual/Introduction_multi_processor.html > and the following sections). > > > > I have conducted consistency tests on these data. I guess I'd like to > know > > what the threshold might be for something that has a Tc = 5 ns. > > The consistency tests are independent of the correlation time. You > must however input relaxation rates and not times. There is a good > description of what problems to look for in the manual > ( > http://www.nmr-relax.com/manual/Consistency_testing_script_mode_visualisation_data_output.html > , > though the PDF is of higher quality). Also have a look at Sebastien > Morin's paper on the subject (he implemented the consistency testing > in relax): > > - Morin, S. and Gagn ́e, S. (2009a). Simple tests for the > validation of multiple field spin relaxation data. J. Biomol. NMR, 45, > 361–372. (http://dx.doi.org/10.1007/s10858-009-9381-4). > > > > My protein behaves as a single domain to the best of my knowledge. It is > > actually a tethered complex between two proteins but they bind each > other. The > > dynamic region near the C-terminus is the glycine-serine loop that > connects > > the two entities. I don't believe it comes apart very often as one of the > > proteins precipitates if left on its own. > > > > There appears to be no concentration-dependent dimerisation. The HSQC > spectra > > of the protein at 0.04 and 0.6 mM look identical. > > From the data, it appears as if everything should be fine, apart from > the NOE errors. However I can see that one spin has an NOE value of > > 1. This is not physically possible, so you should probably deselect > this spin in the analysis and look back at the spectra to see why this > is the case. > > > > The relaxation data for the 600 and 800 MHz exp are attached. The S2 has > also > > been graphed. Does anyone know why my S2 parameters are the opposite to > what > > I'm expecting? > > As I mentioned above, the S2 values look exactly as I would expect if > times and not rates are feed into the program. I hope this > information helps. > > Regards, > > Edward > > _______________________________________________ > relax (http://www.nmr-relax.com) > > This is the relax-users mailing list > [email protected] > > To unsubscribe from this list, get a password > reminder, or change your subscription options, > visit the list information page at > https://mail.gna.org/listinfo/relax-users >
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