Sequential assignment (2)

Finding a sequential link

In order to illustrate the sequential assignment procedure using the setup in the previous section, we will start from a spin system that we previously identified as an Alanine, spin system {23}. In {Spin System Table} scroll down to spin system 23 ({23}Ala) and select it. The HCN and HCN(2) windows will now focus on respectively the Query spin system ({23}) and the spin systems that match the sequential i-1 connectivities of {23}:

The matched peak positions in {Spin System Table} now show 4 potential matches in window HCN(2), for this sequential link: Spin systems {68}, {71}, {45} and {52} in order of their respective score under |Total Peak Score|. Selection of the correct sequential spin system can be done using the rules:

• • The type of match. I.e. the query sequential connectivity should match with an intra residual connectivity. This information is obtained from the combination of the CBCAcoNH and HNCACB spectra

  • The number of matches. The higher, the better.

  • The score of the matches. The higher, the better.

In this case it is easy to verify that the only possible match is spin system {71}, of which both intra residual peaks match the sequential peaks of spin system {23}. {68} is discarded because the matching peaks are both sequential; {45} and {52} are discarded because the intra residual CA's clearly doesn't match the sequential CA peak of {23}. We now assign {71} as the i-1 sequential neighbour of {23} by selecting it and pressing [Set Seq Link]. The list of spin systems now has {71} as a neighbour of {23}Ala:

Mapping sequentially linked spin systems to the sequence

After sequentially linking a number of spin systems, the linked spin systems have to be mapped to a location in the protein sequence. In the vast majority of cases, the mapping of sequentially linked spin systems to the protein sequence requires the identification of the residue types of the spin systems as described previously. The Assignment: Protein Sequence Assignment popup provides the functionality to map a stretch of sequentially connected spin systems in the 'Sequence Locations' and 'Residue Types' tables:

• • 'Residue Types' shows scores for the spin system type matching for a sequence of 5 spin systems, from i-2 to i+2, with the current spin system set to i.

  • 'Sequence Locations' ranks stretches of 5 sequential residues in the protein sequence according to a score calculated from the sequentially linked residue types.

As described in Identifying spin system types, a reliable spin system typing requires the assignment of as many as possible atom types in the spin system, of which the most important are the CA and side chain carbons. So far, we have only done this for 2 spin systems, Ala{23} and Ile(42}, whereas the rest were left unassigned. Here we will illustrate how sequential linking, side chain assignment and residue typing are integrated in an efficient assignment procedure that can be used for complete assignment of a protein.

We start again from Ala{23}, and its sequential i-1 neighbour {71}. Ala{23} has its CA and CB shifts assigned. {71} has no side chain atom assignments yet, but based on the pattern it is already scored as most likely being a Valine (verify!). The highest ranking sequence location is residue 33-37, which as you will see below is the correct match, even after making only a single sequential link and with very limited residue type information. Now using the strip for {71} in window HCN(2), identify and assign the CA and CB resonances of {71} and add them this spin system:

• • Select correct peak

• R: Assign: HNCACB....

  • Set atom type

  • Set same spin system

Distinguish sequential and intra residual using the CBCAcoNH spectrum and HNCACB spectra. After assigning the CA and CB of this spin system, the residue type scores and sequence matches change and lower the uncertainty in the assignment:

After assigning the rest of the side chain resonances using the procedures to assign side chain resonances, {71} can be assigned unambiguously to a Valine (verify!). To continue the sequential assignment from Val{71}, click on [Goto i-1], and verify that {64} is the best matching i-1 neighbour of {71} (note that you may have to change the tolerance):

After the complete assignment of the side chain resonances of {64} you should get:

The first predicted Lysine showed to be and Glutamate which spin system {64} here has been assigned to. This Glutamate was revealed under the side chain assignment. This illustrates an example of how it can be necessary to work in parallel with the sequential assignment and the side chain assignment. When you are certain of a sequential assignment, select the correct match from 'Sequence Locations', and click [Assign Selected].

The procedure can now be repeated for the rest of the spin systems to obtain further backbone and side chain assignments. It is clear that adding more information and sequential links increases the reliability of the sequential assignment. In particular, it is important to pay attention to the location of breaks in the sequence from the occurrence of Prolines, and the location of Glycines, Alanines, Serines and Threonines. These residues are good markers for correct sequential assignments.

The project so far has been saved as NapD_7.

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