Sequential assignment (1)

This section describes the basic steps for how to use the built in functionality to link sequential residues. Subsequently I will describe how complete assignments can be reliably obtained by integrating the sequential linking with side chain assignments and residue typing.

Preparation

Before starting the sequential assignment we will create a duplication of one of the windows. The reasons for this will become clear when we set the parameters for the sequential assignment procedures below. To do so, we will first duplicate (clone) the window HCN, and then name the cloned window "HCN(2)":

• • In the HCN window, use R: Window: Clone

  • Edit the name of the window using the Window: Windows popup: M: Window: Windows, set it to "HCN(2)".

Linking sequential spin systems or residues is done using Assignment: Protein Sequence Assignment, which can be accessed through M: Assignment: Protein Sequence Assignment:

BTW, how can we use HNcaHA and HBHAcbcacoNH in this panel??

The popup consists of 3 tabs, the {Windows & Spectra}, {Spin System Table} and {Options} tabs. The important parts of the {Windows & Spectra} tab are the selection of the query windows and match windows. Initially we will search for i-1 neighbours, which means that we will use:

• • Query spectra: CBCAcoNH

  • Matches spectra: HNCACB

We will display the results of the matching in 2 HCN window:

• • Under 'Query Windows and Spectra' in "13C Window:" set HCN

  • Under 'Match Windows and Spectra' in "13C Window:" set HCN(2)

Note that throughout the analysis one can toggle the Query and Matches spectra, to search for i+1 neighbours. The choice to use the 2 duplicated windows for the Query and Matches is a personal one.

Before we start the procedure of searching for sequential neighbours and linking them, we need to adapt the Peak Matching parameters:

• • Set the tolerances to 0.3 initially, both for CBCAcoNH and HNCACB. This is the tolerance used for finding neighbour matches. Depending on the situation, e.g. in the case of overlapping peaks, the tolerance can be varied to find the correct matches.

Now enter {Options} to set some parameter settings for the matching routines

• • Set the "13C Focus width (ppm:)" to 30 ppm (or any other value that is convenient). This parameter has effect on the display of the matching peaks in the spectra.

  • Uncheck the box that refers to "Focus 13C matches".

  • Leave the other parameters to the default values.

Now enter the {Spin System Table}:

The Spin System Table displays all the existing spin systems, sequential matches etcetera, and is used to update the information from the matching procedures and make sequential assignments. For convenient analysis, it is recommended to line up the several windows on your screen:

Previous: Assigning side chain resonances

Next: Sequential assignment (2)