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Today's Topics:
1. Re: Temperature calibration: methanol- vs protein-based,
(Martin Ballaschk)
2. Re: How to get model free parameters from output files
(Edward d'Auvergne)
3. Re: Temperature calibration: methanol- vs protein-based,
(Edward d'Auvergne)
----------------------------------------------------------------------
Message: 1
Date: Fri, 11 Jan 2013 10:35:58 +0100
From: Martin Ballaschk <ballaschk@xxxxxxxxxxxxx>
To: "relax-users@xxxxxxx" <relax-users@xxxxxxx>
Subject: Re: Temperature calibration: methanol- vs protein-based,
Message-ID: <02A559F2-79A9-4E7C-A5E4-0D09DF8F048A@xxxxxxxxxxxxx>
Content-Type: text/plain; charset=windows-1252
Hi Edward!
On 08.01.2013, at 10:31, "Edward d'Auvergne" <edward@xxxxxxxxxxxxx> wrote:
For the temperature issues, maybe it is worth talking to the Bruker
people.
I will do that. I should have done it earlier, I guess.
Every spectrometer behaves differently with respect to temperature, so
temperature calibration is essential for any serious dynamics study.
It is incorrect and dangerous to assume that the VT temperature
setting is the same as the sample temperature.
We're aware of that, that's why we calibrate the spectrometers
regularly with the help of our standard methanol sample. Our method
just seems not that accurate as I thought. I will try ethylene glycol.
https://gna.org/patch/download.php?file_id=16912
https://gna.org/patch/download.php?file_id=16913
From your spectra (the above file links), it looks suspiciously as
though you have an exchange process occurring (though it may not be
the case). Have you measured relaxation dispersion data for the
system?
All the dynamics stuff is pretty new to us and we just started
implementing the pulse schemes for the dispersion experiments. So,
no, we didn't yet measured relaxation dispersion, but it is
definitely on the schedule.
What makes you think there is exchange? In the pictures above only
the magenta/pink spectrum is a bit noisy (just above base plane
level) and one peak seems a bit displaced (~ 9.8/132.3 ppm, it's a
Methionine). This residue is not exposed to the solvent and also
tightly bound in a beta-sheet. The effect apparently is
field-dependend, maybe due to ring currents from a Histidine which
are found close to the Met?
But indeed ? during the assignments of the proteins I noticed strips
of disappearing/broadened signals in my spectra. I have four
different complexes, not all of them show the same behavior in this
respect. Some of them pretty much have a complete set of signals,
except for loop regions. At least one protein has clearly a reduced
set of signals. Also, some ?double? peaks appear. I don't yet have a
coherent picture of the dynamics of my proteins, but this is why we
started the project in the first place ;) Spin relaxation seemed the
best method to begin with, but there are still a lot of things one
has to take into account, one of them apparently perfectly
temperature-calibrated spectrometers.
For me, the spectra are perfectly identical. The only differences are
due to spectral noise shifting the peak randomly in height and
chemical shift in all directions equally.
Same here after I adjusted for the temperature via my
TROSY/protein-based method.
I would recommend ethylene glycol. Have a look at the published
literature on NMR temperature calibration and see what is best. Maybe
you need to modify the phases on your pulses to preserve the ethylene
glycol magnetisation but destroying the water and protein signals, and
then use the data from the recorded 2D for your calibrations. This
would avoid quick cooling between the experiments.
That's a good idea. I will look into this.
Thanks a lot,
Martin
------------------------------
Message: 2
Date: Fri, 11 Jan 2013 11:02:18 +0100
From: "Edward d'Auvergne" <edward@xxxxxxxxxxxxx>
To: Martin Ballaschk <ballaschk@xxxxxxxxxxxxx>, Shantanu Bhattacharyya
<esesbee83@xxxxxxxxx>
Cc: "relax-users@xxxxxxx" <relax-users@xxxxxxx>
Subject: Re: How to get model free parameters from output files
Message-ID:
<CAED9pY_XyVFyOskEY=iXx866ddLGU38iKktGjF7ts1rQJ75sDw@xxxxxxxxxxxxxx>
Content-Type: text/plain; charset=ISO-8859-1
Hi,
For reference, in the future can I ask you not to attach files to
public mailing list messages. Cheers! Attachments cause a large
strain on the open source infrastructure behind relax. If you need to
attach files, then this can be performed using the relax trackers
(bugs, tasks, support request, patch, etc) at
http://gna.org/projects/relax.
For the results, note that what you have calculated is only a small
fraction of a full model-free analysis. Firstly, how did you
determine the initial diffusion tensor?
Your next step will be to optimise the diffusion tensor together with
all model-free parameters, i.e. the 'global' model. Note that the
starting diffusion tensor will be different to the final diffusion
tensor. So then you need to repeat everything using the final
optimised diffusion tensor as the initial diffusion tensor. Using a
different diffusion tensor at the start (i.e. the final optimised
tensor) will give you different final results. After repeating, you
can then compare the optimised diffusion tensors again and see that
they are different, and that the model-free results are also
different. You have to repeat this up to 20 times (sometimes much
less, other times much more) until the chi-squared value is identical
between two rounds (to the last decimal place), the model-free models
and parameters are identical, and the diffusion tensor parameters are
identical. Please see my 2007 review
(http://dx.doi.org/10.1039/b702202f), and 2008b paper
(http://dx.doi.org/10.1007/s10858-007-9213-3) for details about this
iterative procedure. There are other useful details in my other
papers (see http://www.nmr-relax.com/refs.html). And the model-free
chapter of the relax manual also goes into a lot of detail about this
iterative procedure (http://www.nmr-relax.com/manual/).
You will then need to complete this iterative procedure separately for
the spherical, oblate and prolate spheroidal, and elliptical diffusion
tensors. You then need to perform model-selection between these
global models (AIC is useful here as these are non-nested models and
hence F-tests are not applicable). This is all part of current and
past model-free protocols.
Also note that there can be a circular pattern in your iterative
optimisation. This combined optimisation/model selection problem can
spin in a perpetual loop around the universal solution (the solution
in the universal set, which is the combination of all global models of
the diffusion tensor plus all model-free models and the optimisation
spaces of each global model). For more details, see my 2007 paper.
So you will need to catch this circular pattern and terminate
optimisation when it is reached. For reference, the
dauvergne_protocol auto-analysis built into relax performs such checks
(auto_analysis/dauvergne_protocol.py).
Only once all of this is completed will you be ready to use the
value.write and grace.write user functions for data visualisation.
Until then there is a lot of literature to catch up on ;)
Regards,
Edward
On 8 January 2013 13:57, <mengjun.xue@xxxxxxxxxxxxxxxxxxxx> wrote:
Dear Dr. Edward d'Auvergne,
I have tried to run mf_multimodel.py under relax-2.1.2 for analysis of
single field data (demo data ubq), it seems it work well. I also run
modsel.py, the model M5 is good for most residues. I would like to ask you
about the output file, the model free parameters calculated (for example,
M5) are dispersed in the text of output file (resultS.bz2, or log file), how
to get the model free parameters from these output files, so that the
parameters can be input to other software,for exapmple, origin or sigmaplot.
Attached please find the log file or results.bz2 for M5. Thank you.
With best regards,
Mengjun Xue
------------------------------
Message: 3
Date: Fri, 11 Jan 2013 11:16:08 +0100
From: "Edward d'Auvergne" <edward.dauvergne@xxxxxxxxx>
To: Martin Ballaschk <ballaschk@xxxxxxxxxxxxx>
Cc: "relax-users@xxxxxxx" <relax-users@xxxxxxx>
Subject: Re: Temperature calibration: methanol- vs protein-based,
Message-ID:
<CAED9pY_00NX31AJMC8j00bsHDZEE4wsT_SiPMD5807k5qyTMrA@xxxxxxxxxxxxxx>
Content-Type: text/plain; charset=windows-1252
Hi,
Every spectrometer behaves differently with respect to temperature, so
temperature calibration is essential for any serious dynamics study.
It is incorrect and dangerous to assume that the VT temperature
setting is the same as the sample temperature.
We're aware of that, that's why we calibrate the spectrometers
regularly with the help of our standard methanol sample. Our method
just seems not that accurate as I thought. I will try ethylene
glycol.
Note that different experiments add different amounts of power into
the probe. So this calibration is ok for most standard experiments
but is not suitable for high powered experiments (such as with intense
spin lock periods, CPMG blocks, etc.) when temperature is important
for you.
https://gna.org/patch/download.php?file_id=16912
https://gna.org/patch/download.php?file_id=16913
From your spectra (the above file links), it looks suspiciously as
though you have an exchange process occurring (though it may not be
the case). Have you measured relaxation dispersion data for the
system?
All the dynamics stuff is pretty new to us and we just started
implementing the pulse schemes for the dispersion experiments. So,
no, we didn't yet measured relaxation dispersion, but it is
definitely on the schedule.
What makes you think there is exchange? In the pictures above only
the magenta/pink spectrum is a bit noisy (just above base plane
level) and one peak seems a bit displaced (~ 9.8/132.3 ppm, it's a
Methionine). This residue is not exposed to the solvent and also
tightly bound in a beta-sheet. The effect apparently is
field-dependend, maybe due to ring currents from a Histidine which
are found close to the Met?
But indeed ? during the assignments of the proteins I noticed
strips of disappearing/broadened signals in my spectra. I have four
different complexes, not all of them show the same behavior in this
respect. Some of them pretty much have a complete set of signals,
except for loop regions. At least one protein has clearly a reduced
set of signals. Also, some ?double? peaks appear. I don't yet have
a coherent picture of the dynamics of my proteins, but this is why
we started the project in the first place ;) Spin relaxation seemed
the best method to begin with, but there are still a lot of things
one has to take into account, one of them apparently perfectly
temperature-calibrated spectrometers.
Just from the peak shapes, the differences in peak intensities, and
the temperature shifts gives the impression of an exchange process.
But this is just a hunch. This is also the Trp indole NH region of
the spectra where I would expect all peaks to be equally intense due
to nanosecond ring flips in the core and surface of the protein
affecting the relaxation properties of the spin systems, resulting in
strong peaks as you would expect from mobile bb sections of the
protein. But you would need to confirm this. And if exchange is
involved, then temperature is even more important ;) From the parts
of the spectra you show, it looks like both standard relaxation and
relaxation dispersion could possibly produce quite interesting
results.
Regards,
Edward
------------------------------
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Re: relax-users Digest, Vol 78, Issue 5