CHESPA Projection Analysis

CHESPA Projection Analysis

Three states are required for CHESPA, the inactive reference state (state 1), the active reference state (state 2) and the perturbation state (state 3). The reference vector is computed from the inactive to the active reference states (vector 1 to 2). The perturbation vector can be originated from either the inactive reference state (vector 1 to 3) or the active reference state (vector 2 to 3), depending on whether the perturbation state is relative the inactive or active state. It is recommended to originate the perturbation vector from state 1 when probing an activator and from state 2 when probing an inactivator. However, both vectors can be analyzed.

Generally, a correction factor for the N dimension is required to avoid skewing the analysis towards ¹⁵N ppm changes. We recommend the ratio of 0.2. Similarly, a minimum chemical shift difference of 0.05 ppm is defaulted for both vectors to filter out possibly insignificant chemical shift differences. Residues with chemical shift difference below the set ppm cutoff in either vector are excluded from cosθ and fractional projection analyses. Additionally, an option to filter out overlapping peaks is also implemented to avoid errors arising from poorly resolved peaks. For this purpose, an inter-peak chemical shift difference can be set or the average line width of integrated peaks can be used.

Click “Analyze” once the vectors and parameters are defined. The results will be automatically saved under the folder of “CHESPA” in the system default Sparky folder. You can use the “Save” button to rename the results for your own naming convention and use the “Load” button to reload previous data. (Note: the spectra involved in the analysis need to be open in order to load CHESPA data). Once the data is loaded, you can click “plot” to view the CHESPA results. Four plots will be shown against residue numbers. The compounded chemical shift (CCS) difference plots of the reference and perturbation vectors, the cosθ and the fractional projection from the perturbation vector onto the reference vector. The compound chemical shifts, cosθ and fractional projection of individual residues can also be viewed once loaded. By double clicking each individual residue, corresponding peaks in the spectra involved in the analysis can be shown.

The CHESPA results can also be plotted onto a protein structure on PyMOL. You can choose a pdb file of your choice after clicking the “PyMOL” button. CHESCA-SPARKY will create a pdb file in your data folder called “chesca.pdb” which will realign the input pdb file with the assignments from the spectra. A PyMOL command line is copied to the clipboard as well. If the PyMOL program does not start automatically, you can simply start PyMOL and paste the command line. To show individual residues, select the residues you want to see in the result window and type “@p” in the PyMOL command-line to update the structure accordingly.