Correlated Peak Analysis
RunAbout is a tool combining visual display with computational analysis to obtain the complete chemical shift assignments of proteins. It is explicitly designed not to be a "One Button" tool, but rather to facilitate much of the process with computer power and let the user see and interact with the data. Central to the data visualization is the correlated display of related experiments so the user can integrate the data from multiple experiments in one display. Shown here is RunAbout in the Peak Display mode which is used to first get a visual assessment of the quality of the datasets, the peak picking and peak alignment.
A variety of computations can be initiated through this interface. For example, you can automatically align multiple triple resonance experiments, filter out artifactual peaks based on alignment with a reference list, trim weak peaks based on the expected number of peaks for each residue, and cluster peaks based on their common amide hydrogen and nitrogen frequencies.
Clusters are groups of peaks that share a common amide proton. They might be intra or inter-residue peaks representing CA, CB or C atoms. After computationally clustering peaks you can use the cluster editor to interactively view and change clusters. The cluster details window shows you all the peaks you would expect for the cluster, the peaks actually found, their chemical shift similarity, and the expected amino acid types based on the known chemical shift distributions. If you have prior knowledge of the cluster's amino acid type (perhaps from labeling experiments) you can click the check boxes to restrict the assignment to those types.
RunAbout CA-CB graph
This graph takes the assembled cluster information and displays it as if you had a 2D CA-CB experiment. Each pair of adjacent residues should have two overlapping dots, blue and red for the intra and inter-residue peaks. Confirmed overlapped clusters are indicated by the presence of a black dot. The graph is interactive so you can zoom in, and confirm cluster pairs. Rectangular regions indicate the typical chemical shift ranges for the different amino acids.
Runabout Link Editor
Overlapping clusters corresponding to adjacent residues in the sequence are built up into fragments using the Link Editor. In this mode you not only see the actual aligned spectra, but a variety of quantitative information relating potentially linkable clusters. This includes information indicating whether the fragment that would be formed by linking the clusters could match to a position in the sequence. Fragments can be automatically extended if the quantitative scores are high enough.
HCCH Tocsy Mode
RunAbout can use HCCH Tocsy information to assign side chain atoms. Peaks are clustered based on the HC shifts and then presented as possible matches to the existing backbone assignment data. A scan through the raw dataset data is done to find carbon shifts that have a proton intensity profile matching the that found for the HA or HB atoms. The atom table in this mode displays all the possible atoms for the current residue and the color and numerical score indicate the probability that the selected peak could be assigned to that atom type. In this mode you can also display a NH-NOESY experiment to provide confirmatory data for the assignment.
Side-chain Tocsy experiments that are detected through the amide proton can also be used in the analysis. Expected shift positions for protons and carbons for the current residue are displayed directly on the spectra and can be assigned with a few clicks of the mouse.