Hi Edward!
On 08.01.2013, at 10:31, "Edward d'Auvergne" <[email protected]> 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 _______________________________________________ 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
