The right-hand portion of RunAbout’s Edit Clusters display presents your spectra in a way that aligns peaks from different experiments, thus enabling you to assess whether they truly belong in the same clusters. These can be arranged however you want by configuring the “parameters” panel, but this default configuration works well for most circumstances. Every cluster has a single pair of 1H and 15N frequencies, so every peak in this display shares these values. The left two columns display spectra showing only (i)–to–(i-1) connectivity: the HNcocaCB, (shown here; alternatively, you can show the HNcoCACB to include CA peaks), the HNcoCA, and the HNCO. Of these two, the leftmost column’s panels’ X-axes correspond to 15N frequency, and others correspond to 1HN frequency. The right two columns display spectra showing both (i)–to–(i-1) and (i)–to–(i) connectivities: the HNCACB (in which CB’s appear negative and CA’s appear positive), the HNCA, and the HNcaCO. The rightmost column’s X-axis corresponds to 15N frequency, and the other column’s to 1HN.
For most residues, you observe that the three different types of (i)–to–(i-1) 13C frequencies each appears as four peaks aligned horizontally. Some spectra, such as the HNCO and HNcaCO shown here, may have been taken with different 13C spectral widths, in which case the peaks will not exactly align visually, but horizontal cursors will still correspond to the same frequencies in different spectra. You also observe that the three types of (i)–to–(i) 13C connectivities share 13C frequencies. When both residues i and i-1 are not glycine or proline, you should expect to observe 11 peaks in the cluster (13 if you used the HNcoCACB or CBCAcoNH instead of the HNcocaCB). The number of peaks you would expect to see in a cluster, depending on residue type, is displayed in Table 1.
Table 1. Numbers of peaks expected in RunAbout clusters. Residue i-1 Residue i Expected peaks in cluster (w/ HNcocaCB/CBcacoNH) Expected peaks in cluster (w/ HNcoCACB/CBCAcoNH) Non-Gly Non-Pro, Non-Gly 11 12 Gly Non-Pro, Non-Gly 9 10 Non-Pro, Non-Gly Gly 10 11 Non-Pro, Non-Gly Pro 0 0