> Description of the problem:
> I have a set of triple resonance experiments and a 15N-HSQC. I
> initially picked the peaks from all the spectra anonymously. Given
> the set of typical triple resonance spectra, I was able to connect
> the peaks from the same residue and its preceding residue to their
> corresponding amide peak (NH) in the 15N-HSQC, i.e., establish i and
> i-1 connectivity of the anonymously numbered spin systems.
> Eg., HNCA: {5}N[10],{5}N[11],{5}CA[200] (added to the spin system
> using 'R:' menu); {5}N[10],{5}N[11],{6}CA[203] (added to the
> sequential spin system using 'R:' menu)
> HNcoCA: {5}N[10],{5}N[11],{6}CA[203]
>
> Expected Results:
> You expect the inter-residual peak to get the corresponding
> assignment automatically. However, it does not. Exceptions are
> spectra like HNCA which have both i and i-1 information, where it
> seems to work fine. It remains anonymous in spectra that give only
> i-1 information like HNcoCA, HNCO.
Having looked through the peak list and spin system tables it is clear
that all of the spin systems in the project are a complete and utter mess.
There is most likely nothing wrong with Analysis with regards to spreading
assignments along sequentially connected segments.
There are duplicate spin systems for almost every residue. Each HNCA peak
has a spin system, assigned for the most part, but the CA resonances in
the HNcoCA experiment are not linked to these, they are in some other
anonymous spin system.
If the sequential connectivity is set for the HNCA peaks then this will
not affect the HNcoCA linked anonymous spin systems as they are separate
entities. Also, if the sequential connectivity for these anonymous HNcoCA
linked spin systems is set at i-1 relative to the amide/root spin system,
before the root spin system has an assignment, then all you end up with is
lots of pairs of sequentially connected spin systems not a continuous
chain.
The idea is that you should assign the HNCA and HNcoCA matching peaks to
the same carbon resonance (i.e. in the same spin system). I suspect that
liberal use of "Set sequential spin systems" without the HNcoCA being
connected to the same resonance as HNCA has caused the mess.
Analysis has no innate way of knowing which HNCA spin system you meant to
connect an HNcoCA peak to. You have to make a choice on the basis of
matching peak positions and make the resonance connection yourself, in the
same way that you'd do any other assignment.
"Set sequential spin systems" should be used in the manner described in
the extended Analysis tutorial.
T.
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Dr Tim Stevens Email: [log in to unmask]
Department of Biochemistry [log in to unmask]
University of Cambridge Phone: +44 1223 766022 (office)
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