Hi Maddy,
The consistency testing analysis is more a qualitative one than a
quantitative one. That means that the decision for inclusion or not of a
dataset is helped with the test, but that no real cutoff has been proposed.
In my experience, I would say that a J(0) ratio of more or less than 5%
off (>1.05 or <0.95) should point to problems. However, there could be
cases where a proteins has widespread slow motions affecting R2 globally...
Another point is that these tests should be used to judge on the quality
of an entire dataset, rather than quality of data for a single residue.
This is because the use of consistency tests was developed to remove the
influence of systematic bias of datasets. Hence, if a dataset average
J(0) ratio is outside, let's say, the limits discussed above, then it
should potentially be entirely removed, unless only a subset of
residues, for example with slow motions (i.e. with Rex) cause this
skewed average.
If you wish, you could send me the results of your analysis, potentially
in the form of both a correlation plot of J(0) values for different
fields, as well as an histogram plot of the J(0) ratios (please see
figures 1 and 2 of Morin & Gagné (2009) Simple tests for the validation
of multiple field spin relaxation data. Journal of biomolecular NMR, 45:
361–372).
All the best,
Séb :)
On 11-07-06 3:05 PM, Maddy Strickland wrote:
Hi Edward,
Yes - what we did was made sure all the R2 experiments were mixed up so
that you wouldn't have any problem with sample heating (so 10 ms followed
by 1000 ms etc.). We also did two datasets for each of T1 and T2
experiment, alternating T1 and T2 datasets in order to compensate for
sample heating. Temperature didn't go up or anything. I don't know about
the calibration, I think we did this on the first protein we did and it
was fine... Would you recommend using the methanol then, would you say you
see much of a difference?
I did do the consistency testing, wasn't really sure which results to
throw away, do you have a margin within which you would expect most
experiments? I just left out those results which were really off.
I did just redo the peak picking for this protein and actually the
results are coming out fine now, it didn't like just having the same point
picked in each spectrum, it needed the actual peaks picked, no matter how
small. Also I've just noticed in the manual it says to do the RMSD of an
area which doesn't have peaks in - I didn't realise this, I thought it was
over the whole spectrum, so I've changed that now too - although I imagine
it won't have an effect on the actual results, just their errors?
Maddy
-----------------------------------------------------------------------------------------
Hi Maddy,
It could well be that there is an issue with the data. I was
wondering if you'd seen Sebastian Morin's consistency testing work
before? This is part of relax and can be used to check the data. See
http://www.nmr-relax.com/refs.html#Morin09. Considering this is the
R2, there could have been sample heating problems. Are these
experiments temperature compensated? And did you calibrate your
temperatures on MeOH using a single scan directly after a partial R2
experiment completed?
Regards,
Edward
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Sébastien Morin, Ph.D.
Postdoctoral Fellow, S. Grzesiek NMR Laboratory
Department of Structural Biology
Biozentrum, Universität Basel
Klingelbergstrasse 70
4056 Basel
Switzerland
Re: Temperature calibration T2