The Chemical Shift Index is a measure of the deviation of the actual chemical shift assigned to an atom, and a random coil (or other standard) value for an atom of the same type and residue. Since the sign and magnitude of these deviations correlate with the secondary structure type that the residue is found in, this provides a way to get a measure of the secondary structure profile of the molecule from just the chemical shift assignments.
The first step of doing a CSI calculation is determining which atom types are going to be used in the calculation, and displayed in the graphical viewer. You can set this in the CSI Calc and CSI Display rows of the Atoms tab. More then one type of atom may be selected for the calculation of the CSI and for displaying the results. It is also possible to calculate the CSI of more then one atom type and display the result of only one atom type at a time. The result of the CSI calculation can be displayed using the actual differences in ppm by selecting the Value checkbox in the Display tab. Selecting Threshold will show the results using the index values +1, 0 and -1. In both cases a positive value/threshold will be red, a negative value/threshold will be blue and a zero value/threshold will be gray. After selecting the settings click on the Apply button. This will update the sequence display window (see figure 5 ).
Proper calculation of the CSI values requires choosing appropriate reference data and solvent conditions. Thanks to various user contributions, NMRViewJ includes a variety of reference datasets representing values collected under different conditions, including denaturants. You can select the Reference dataset in the Reference tab. Clicking the Infobutton will bring up a new window with some information about the selected database
In the Reference tab you can also select which Chemical Shift Reference compound (DSS, TMS, etc.) was used when collecting and assigning your datasets. If the standard used in the experiments differs from the standard used to obtain the values of the database the CSI program will automatically include a correction factor if necessary.
The next tab, Conditions, can be used to enter the information about the pH and temperature used to obtain the chemical shifts. If the data was obtained using deuterated protein and the chemical shift data has not been corrected select the Deuterated Protein option and the CSI program will correct the carbon chemical shift data automatically. The last set of parameters refer to special experimental conditions which may have an effect on the observed chemical shift. The CSI program can correct for the effect of cosolvents like TFE, acetonitril, urea or guanidinium HCL on the chemical shift assuming a linear correlation for the correction factor in relation to the concentration of the cosolvent. When the chemical shifts are obtained using the TROSY experiments and the experimental chemical shifts have not been corrected the CSI program will correct for this when the TROSY options is selected. The CSI may also be used to check the assignments. The CSI program will check the assignments within a certain tolerance if the Check Assignment option is selected. The tolerance is calculated using the cutoff value used in the selected database multiplied by a value which is set by the user.
When all parameters are set click on the button to start the CSI calculations. The results will be shown automatically in the sequence display window using the settings selected in the Option window. In this figure the HA, CA, CB and C atoms are used for both CSI calculation and display, and the secondary structure cartoon is turned on. Since Threshold mode is on, the graphical CSI display of circles shows red circles at the top (for shifts consistent with helical structure, blue circles at the bottom (for shifts consistent with sheet structure, and gray circles at the center (for intermediary shifts) of the plot range. The coloring of the top CON row of atoms is by the secondary structure prediction based on the concensus of all the atom types, and with contributions over a three (sheets) or four (helices) residue window. The cartoon display showing sheet or helical regions is based on the filtered, concensus CSI values.
When the Value, rather than Threshold setting is turned on the displayed circles are plotted relative to the maximum absolute delta for that atom type in the current data. In this case, be aware that some atom types (e.g. CB) will have their shifts going in the opposite direction to the way they move in Threshold mode.
If the "Print ascii files" option is selected in the Options window the following files will be created in the directory where NMRViewJ has been started. ATOMTYPE.csi.REF : file containing the data for the CSI per residue. ATOMTYPE.ppm.REF: file containing the difference (in ppm) between the chemical shift data and the random coil values per residue. ATOMTYPE.csi.as.REF: file containing the observed chemical shifts per residue and a flag indicating if the values are within the assignment tolerance (0) or outside the assignment tolerance (1) or no assignment tolerance was selected (-1). consensus.txt: Only when three or more different atom types are used in the CSI calculation will this file be created. It contains information about the consensus secondary structure elements between the different selected atom types.