Hi Nav, I had similar problems in the past.
The inconsistetncy has to come from somewhere. For me, it was the temperature difference between different magnets. After I figured out that we calibrated our spectrometers the wrong way, I finally got consistent data. Just try to superimpose two high-resolution spectra from your two fields. They should be identical. If you can see peaks that are not 99% on top of each other, I would recommend take a long hard stare at temperature control. How do you control your temperature? I found neat methanol did not work with our spectrometers with cryoprobes, see also [1]. We use d4-methanol now, the impurities give enough signal for a proper temperature calibration (between magnets, and between experiments). Another issue may be TROSY-based sequences, used on deuterated systems and cryoprobes. As described by Nils Lakomek et al, that can be the source for a lot of annoying artifacts. [2] Edward will have additional ideas, I guess. Cheers Martin [1] Lakomek N-A, Ying J, Bax A (2012) Measurement of 15N relaxation rates in perdeuterated proteins by TROSY-based methods. J Biomol NMR 53: 209–221. doi:10.1007/s10858-012-9626-5. [2] Findeisen M, Brand T, Berger S (2007) A1H-NMR thermometer suitable for cryoprobes. Magn Reson Chem 45: 175–178. doi:10.1002/mrc.1941. On 26.04.2013, at 14:31, Navratna Vajpai <[email protected]> wrote: > Hi Edward and other members in the group, > > Just briefly mentioning my concern: I have acquired 15N-backbone relaxation > data on a protein kinase on two different fields (600 MHz and 800 MHz). In > the beginning had some difficulties in running your scripts. Following your > suggestions, I looked through the literature and developed some understanding > before running all these scripts in Relax. The scripts seem all working for > the local_tm model. However, for sphere or the spheroid models, it never > converged (the run continued for several days with going upto 64 rounds). On > looking through the next chapter about data consistency, i thought of doing > consistency tests. Tests with J0 checks, suggests inconsistency as described > in the chapter. As i do not have access to the third field, i do not know > which data amongst the two is bad. Experimental parameters or the sample used > were same at both fields. Is there any way to check this without having data > for the third field? > > Do you or someone else has a script which can use data from only single field > and let RELAX do model-free analysis? > > I looked through the mailing list and have seen that this problem has been > asked and discussed several times. I know about TENSOR2 which can do such an > model-free analysis using single field but was wondering if some has found a > fix for the RELAX. > > Many thanks, > Nav > > > > > > > On Tue, Feb 12, 2013 at 2:59 PM, Edward d'Auvergne <[email protected]> > wrote: > Hi Nav, > > Welcome to the relax mailing lists! Please see below: > > > > The situation: > > I have experimental data for R1, R2 and NOE at two fields (600 MHz and 800 > > MHz) on a large protein kinase. As expected, i do not have data for all the > > residues in the protein sequence. on searching through Web, i have found a > > X-ray structure, which also have some parts missing, possibly due to poor > > electron density in those regions. > > This will complicate your analysis, as you don't have orientational > information about your NH vectors! Such information is essential for > the prolate and oblate spheroidal and ellipsoidal diffusion tensors. > You will need to read the relevant literature if this is not clear > (you can find lots of references in the papers linked at > http://www.nmr-relax.com/features.html#primary_refs, especially my > 2008a paper at http://www.nmr-relax.com/refs.html#dAuvergneGooley08a). > > > > I learnt from RELAX that one can create > > spin system solely based on sequence and then attach protons to it or by > > using a pdb structure. > > relax does not currently have an algorithm to automatically place > protons into the 'correct position' in 3D space. This just allows you > to say that protons are attached - hence you will have dipole-dipole > relaxation present. If you have a 3D structure without protons, you > will need to use Molmol, PyMOL, etc to add the missing protons > yourself prior to loading the structure into relax. > > > > For model free analysis possibly, i would need a pdb > > structure (not entirely sure!); as i can see, an example in the manual > > illustrating without the use of the structure (page 103) > > You really need to read more of the literature to understand the > reason why. But you can perform a model-free analysis using the > protocol I developed which is hard-coded into the GUI. But you can > only use the 'local_tm' and 'sphere' models if no 3D data is present. > If this is not clear why, then you have a lot more reading to do ;) > > > > The problem: > > When i tried doing it by creating spin systems using amino acid sequence > > alone, the system never got executed. However, when i started doing it with > > structure as an input., it did run but then gave me an error message for all > > the spins as follows: > > for spins with all six data parameters: > > spin YYY deselected due to absence of any relaxation mechanisms > > This means that you have not specified the relaxation mechanisms. > Note that if you are looking at 15N backbone data - importantly with > no 13C labelling - then two major relaxation mechanisms are present. > These are the dipole-dipole and CSA interactions. You will need to > tell relax that these are active, and what the physics for these > interactions should be. The reason why you have to do this is because > relax can be used for RNA, DNA, or organic molecules. And even in > proteins, this simple 2 mechanism relaxation may not always be the > case. For example 15N bb relaxation with 13C labelling, you have 3 > direct dipole-dipole relaxation mechanisms, and you have to also take > interference into account. Or for natural abundance 13C CO relaxation > where only CSA relaxation is present. relax allows you to handle > these different cases. > > > > and for spins with no data: > > spin YYY deselected due to absence of any data. > > > > the second one is understandable but not sure about the first one . > > Did you follow the tutorial in the relax manual about using the GUI > for model-free analysis, specifically the section on setting up the > relaxation interactions > (http://www.nmr-relax.com/manual/d_Auvergne_protocol_GUI_mode_relaxation_interactio.html)? > > > > To check whether something is wrong with the complete data sets, > > i created new data files for only first two residues with structural > > coordinates extracted for these two residues. In this case, the program > > worked well. > > You can perform a full analysis using the protocol I developed. If > this is not clear what this protocol is, please see my 2007 and 2008 > papers: > > http://www.nmr-relax.com/refs.html#dAuvergneGooley07 > http://www.nmr-relax.com/refs.html#dAuvergneGooley08a > http://www.nmr-relax.com/refs.html#dAuvergneGooley08b > > For residues which have 3D data, you can perform this analysis. For > missing residues, you may have to use the concept of global model > hybridisation: > > http://www.nmr-relax.com/refs.html#Horne07 > > This will allow you to combine the local tm models for residues > without 3D data with the results from the analysis with 3D data. > > > > Questions from me: > > 1) Does that mean the absence of data for certain spins, loaded either from > > sequence or structure, causes this problem? > > No, this is just an indication that you have not set up your active > relaxation mechanisms in relax. > > > > 2) Can i do the whole analysis just by using the sequence. > > Yes, see above. But it would be much better if you use the 3D info > that you already have, assuming that structure is correct. > > > > 3) Does the software actually need minimum six values (R1, R2 and NOE at two > > fields) for this analysis or it can work with >= 3 values? > > Please read my 2007 and 2008b papers about this! > > > > As for some > > residues, i have < 6 data values. I am currently ignoring those residues > > with < 6 data values as i wasn't sure if model free analysis would be able > > to handle that. > > Again, my publications cover this and what the minimum is and why. > But note that model m8, as I have defined it, has 5 parameters. > Therefore you will require minimally 5 data points. > > > > 4) I am still unclear with the initialization of diffusion tensor. In the > > GUI mode the first row asks for The diffusion tensor parameters: > > I have tried to understand what is written in the manual, but i am not sure > > if i understood it correctly. > > This is also discussed in full detail in my 2007 and 2008b papers as > to why my protocol, which is what you are using when accessing the > relax GUI, requires no initial diffusion tensor. These papers also > explain the concept behind this protocol and the inversion of the > problem of simultaneously finding the interlinked global diffusion > tensor and spin specific internal motions. > > > > Would you be able to guide/suggest me on this. Any suggestions from your end > > is highly appreciated. > > One other very useful reference which contains the answer to all your > questions (apart from the missing relaxation interaction setup) is my > PhD thesis which you can find at: > > http://www.nmr-relax.com/features.html#primary_refs > > I hope some of this information helps, but you do have quite some > reading ahead of you! > > 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 -- Martin Ballaschk AG Schmieder Leibniz-Institut für Molekulare Pharmakologie Robert-Rössle-Str. 10 13125 Berlin [email protected] Tel.: +49-30-94793-234/315 Büro: A 1.26 Labor: C 1.10 _______________________________________________ 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
