Hi All, yes there is temperature difference on two different spectrometers. i will re calibrate and possibly re-do the experiments.
Many thanks for all the suggestions. Best, Nav On Tue, Apr 30, 2013 at 4:32 PM, Edward d'Auvergne <[email protected]>wrote: > Hi Nav, > > Martin is spot on here. The temperature control and temperature > calibration has been a topic much discussed on the relax mailing > lists. For example, here are some threads where you can find out more > information, if you wish: > > http://thread.gmane.org/gmane.science.nmr.relax.user/83 > http://thread.gmane.org/gmane.science.nmr.relax.user/273/focus=274 > (this is Chris MacRaild's response to a message by Seb Morin) > http://thread.gmane.org/gmane.science.nmr.relax.user/1121 > http://thread.gmane.org/gmane.science.nmr.relax.user/1419 > http://thread.gmane.org/gmane.science.nmr.relax.user/1368 (this one is > from Martin) > http://thread.gmane.org/gmane.science.nmr.relax.user/1397 (a > continuation of the previous thread) > > You need to click on all the messages in the thread to follow them. > There are many more threads on this area, but I don't have the time to > find them all right now. The following message might be of > significant help for you: > > http://thread.gmane.org/gmane.science.nmr.relax.user/1419/focus=1423 > > Firstly note that if you have a temperature problem, this will not be > solved by looking at a single field strength as it is a problem > between experiments on the same spectrometer. So even if you perform > an analysis with data from a single field strength, the R1 data might > be up to 2 degrees warmer or colder than the R2, and the same problem > will occur to a different degree (or direction) on the second > spectrometer. Any analysis using such data will be meaningless, as > this will have a large effect on the diffusion tensor. Considering > that the diffusion tensor is the major contributor to liquid state > relaxation, the internal dynamics can contribute 20% or less (see the > original model-free papers for these numbers), then any internal > dynamics will be severely distorted, possibly hidden, and artificial > motions will appear. I would recommend you look at this section of > the relax manual for more temperature related details: > > http://www.nmr-relax.com/manual/Temperature_control_calibration.html > > As for relax supporting an analysis at a single field strength, there > is nothing stopping you from performing such analysis. relax is > designed with flexibility in mind, so you can perform your model-free > analysis any way you can imagine. With the minimisation settings, you > can replicate the exact results from Art Palmer's Modelfree, from > Dasha, or from Tensor2. However note that I have not written any > scripts or GUI to handle this situation, as I have no interest to. So > you would need to write the script yourself. I would then recommend > looking at the file 'auto_analyses/dauvergne_protocol.py' to get an > idea of how to implement a full model-free protocol. It is quite big > because of the iterative optimisation of the model-free parameters, > then model elimination, then model selection, and finally diffusion > tensor optimisation, with convergence testing. These steps, even for > single field strength data, need to be iterated until convergence. > This can take up to 15 iterations. You will also need to decide how > to determine your initial diffusion tensor estimate - and relax can > perform this as well. > > But note that you should be aware of of the problems discovered by > Schurr et al., 1994 and Tjandra et al., 1996 of the artificial > motions. For example see: > > http://thread.gmane.org/gmane.science.nmr.relax.user/326/focus=332 > > Korzhnev's review (I don't have the reference at hand) and my paper > (http://dx.doi.org/10.1039/b702202f, this has the Korzhnev reference > in it) cover all of the problems you will encounter and hopefully > convince you that an analysis of single field strength data would only > be useful for perfectly isotropic systems (which is never possible due > to water shell differences around the system) which have no > significant internal motion (but note that if you see no motion with > single field strength data, that does not mean that there is no > motion). > > I hope this helps, > > Edward > > > > > On 26 April 2013 14:48, Martin Ballaschk <[email protected]> wrote: > > 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 > > >
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