Sequence-Specific Assignment

By linking sequential spin systems you will have ended up with a number of long strings of spin systems in their correct sequential order. Now you need to work out which part of the sequence they match up to. In order to do this you need to identify the amino acid type of some of your spin systems. For some amino acid types this is fairly straight forward. Glycine for instance, has a very characteristic Cα and no Cβ chemical shift. Alanine, Serine and Threonine all have very characteristic Cα and Cβ chemical shifts. You can see the distributions of chemical shifts by amino acid type as found in the BMRB within Analysis if you go to Resonance and Reference Chemical Shifts. The following 13C chemical shift table has a visually very appealing way of displaying the average carbon chemical shifts. Using this you can quickly see that Isoleucine, Proline and Valine have lower Cα chemical shifts than all the other amino acids, for example.

Once you have identified (or excluded!) the amino acid type of a few of your spin systems in your linked stretch, you can start to compare this to your protein sequence. You may for instance have a stretch which is S-X-A-X-X-G, where X is, for example, anything other than A, S, G, T or P. If this motif only appears once in your sequence, then you can make a sequence specific assignment. If you like, you can immediately enter this into Analysis. But I tend to wait until I have assigned most of the sequence and am feeling fairly confident with my assignment before doing this, as it makes undoing mistakes easier. To enter your sequence specific assignment in Analysis, simply bring up the Assignment Panel for one of the peaks in one of the spin systems you want to assign. Select a resonance and click Assign {SpinSystem}ATOM[Resonance]. The Atom Browser panel will appear and you now need to select the exact atom which this resonance corresponds to.

Making a sequence specific assignment

This atom will then be shaded in a slightly darker colour in the Atom Browser panel to indicate that it is assigned. But you will find that there are lots of other atoms which will become shaded at the same time, because Analysis is able to assign them based on the assignment you have just made. For instance, if you assign the Cβ in one spin system, then Analysis will automatically be able to sequence-specifically assign the rest of that spin system, too. And if there is another spin system which has been set to be i-1 to this spin system, that is automatically assigned, too. So with one click, you can set the sequence specific assignment of a whole stretch of residues.

Automatic assignment of linked stretches