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 relax-users@gna.org 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