Hi,
The 10 seconds for the NOE seems a little excessive (unless this is an
IDP or very small protein). Have you quantified the time required for
recovery? As for the peak picking and fitting, my experience is that
with the proper temperature control and calibration, then the
following routine is the most accurate for extracting relaxation rates
(this is yet to be published):
- Shift all peaks to their maximum in each spectrum (pc in Sparky for
example).
- Average the peak lists across all spectra for one relaxation data set.
- Measure peak heights using the averaged peak lists.
I would not touch volume integration. One other thing you need to be
very careful with is sample concentration. If you require multiple
samples then you should aim to have identical protein concentrations
(volume does not matter). Slight concentration differences can have a
large effect on the global tumbling of you system, hence the data
cannot be combined. I hope some of this helps.
Regards,
Edward
On 5 February 2013 09:52, Martin Ballaschk <ballaschk@xxxxxxxxxxxxx> wrote:
Hi Edward,
a part of my mail is missing, it seems like I accidentally sent a draft
version.
Although it's maybe not that important, here is the rest of the final
paragraph:
„I don't know if inaccurate or inconsistent data would favor such a
behavior. We now use selective proton pulses in the R1 and NOE-experiments
(like described in [1]), temperature compensation for our R1 pulse
programs, single-scan interleaving for all experiments, accurate
temperature calibration with d4-methanol and automated and hence
reproducable peak picking and fitting routines, and recycle delays of 10s
in the HetNOE. The consistency of the data from two fields was well below
8% even when the temperature was (accidentally) off by 1K.“
Regards,
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.
--
Martin Ballaschk
AG Schmieder
Leibniz-Institut für Molekulare Pharmakologie
Robert-Rössle-Str. 10
13125 Berlin
ballaschk@xxxxxxxxxxxxx
Tel.: +49-30-94793-234/315
Büro: A 1.26
Labor: C 1.10
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Re: m0 models