1. The Y-axis represents the secondary chemical shifts of the nuclei: CA for alpha carbon, CB for beta carbon, HA for alpha proton and CO for carbonyl carbon. Secondary chemical shifts are calculated by subtracting random coil chemical shifts[1] from experimental observed values.
2. The X-axis represents the secondary chemical shift difference between CA and CB.
3. LACS shows the secondary chemical shift relationship between CA (or CB, HA, CO) and CA-CB for a single protein.
4. Points with a positive CA-CB value are fitted to get one line corresponding to the transition from random coil to alpha helix; points with a negative CA-CB value are fitted to get another line corresponding to the transition from random coil to beta sheet.
5. The vertical line is located at the position of CA-CB = 0. Considering the referencing independence of CA-CB, this is the position where an amino acid in random coil state will fall with the highest probability (in the X-dimension).
6. The intersection between the vertical line and the fitted line gives us the position where an amino acid in random coil state will fall with the highest probability in Y-dimension.
7. The existing of two intersection points (with two fitted lines) is due to the limitation of experimental data as well as using a rough linear model. To compensate for these limitations, the random coil highest probability position in the Y-dimension is estimated as the average of the Y-axis of these two intersection points. At this average position, a horizontal line is drawn.
8. An individual nucleus (CA, CB, HA, CO) has the highest probability of being in the random coil state when its secondary chemical shift is zero. Thus the horizontal line represents the possible referencing adjustment needed for the investigated protein.
[1] Wang, L., Eghbalnia, H. R., Bahrami, A., and Markley, J. L. (2005) Linear analysis of carbon-13 chemical shift differences and its application to the detection and correction of errors in referencing and spin system identifications. J. Biomol. NMR 32, 13-22.