Re: Missing data for spin system and compatibility in GUI -- April 26, 2013 - 14:32

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 <edward@xxxxxxxxxxxxx> 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