Having completed the backbone assignments your are now ready to proceed to assigning the side chain atoms. Two of the common sets of experiments for this involve TOCSY experiments that relay magnetization through the atoms of the side chains. The two methods differ in whether whether the magnetization is relayed through the carbonyl carbon and amide nitrogen and ultimately detected on the amide proton, or alternatively remains on the carbons and carbon bound protons.
Because these experiments differ in the best way to present and analyze them, RunAbout provides separate modes for working with them.
To assign TocsyNNH experiments you should first ensure that you have entered the carbon and proton experiments in the parameters tab. Choose the experiment that indirectly detects the carbon atoms in the peak list chooser labeled CCcoNHTocsy. Choose the experiment that indirectly detects the hydrogen atoms in the peak list chooser labeled HCcoNHTocsy. Make sure you check that the pattern and tolerance information for each list. Both experiments should have the amide proton dimension set to i.h (or i.hn) and the amide nitorgen dimension to i.n. The carbon experiment should have the remaining dimensino set to i-1.c* (though any carbon pattern should work) and the proton experiment should have this dimension set to i-1.h*.
In this mode the "Helm" will have controls to step through the assigned residues (so use this tool after assigning the backbone). An atom assignment table is present below the Residue navigation tools. As you move to each residue the atoms present in that residue type will appear, with the carbon and nitorgen atoms in the left column, and any attached hydrogens in the next columns. Next to each name is the current chemical shift assignment for that atom (empty if not assigned). At the beginning of the sidechain assignment process you will typically find that the ca, cb, n and h atoms will have chemical shift values displayed, and the remaining values will be empty. Amide chemical shifts are displayed for your convenience, but are not normally assigned in this module.
The main spectrum window of RunAbout will now be displaying the appropriate spectral regions of the datasets associated with the carbon and proton tocsy experiments you set up in the parameter panel. They should be displayed with the amide proton on the X axis, the amide nitorgen frequency selecting the Z plane, and the sidechain carbon or proton on the Y axis. The amide frequencies are taken from those for the currently displayed residue.
On each carbon and proton Tocsy strip a text symbol will be displayed for each atom type. If the current atom is not assigned, the text background will be yellow, and it will be drawn at the mean chemical shift (on the Y axis) for an atom of that type (and residue type). If the current atom is assigned, the text background will be green, and the label will be positioned at the assigned chemical shift. The text labels will be drawn alternately to the left and right of the center. This is just to minimize the chance they'll appear directly on top of each other.
Make sure the cursor is in selection mode (Use the Cursor menu on RunAbout) and then select a peak in either the carbon or proton tocsy that you're interested in assigning. A number will appear at the right side of each row of the table. This number indicates the probability that the selected peak is of that atom type. If both a carbon and proton peak are selected the probability will be based on the chemical shift distribution of both atom types. The atom names in the table will be colored with a green or red background, based on whether they are an acceptable (above a certain probability) assignment for that peak.
You can assign the selected peak by clicking on the atom label (CB,CG, HB etc.) that is displayed on the spectrum. When assigned, the peak shifts will inserted into the appropriate entry of the table, and the atom assignment table will be updated.
If the selected atom has already been assigned (has a chemical shift value), you will prompted as to whether you want to replace the existing assignment. You can also enter assignments by just typing a chemical shift value into the table.
