Theoretical profiles and fitting from models – PDB2SAS (DENSS) in RAW

RAW has a built-in method to calculate theoretical scattering profiles from atomic models and fit them to experimental data. This method is based on the DENSS PDB2SAS calculator (corresponding to the DENSS command line tool called denss.pdb2mrc.py). The purpose and output of PDB2SAS is similar to that of CRYSOL, but uses a fundamentally different algorithm. PDB2SAS calculates a theoretical scattering profile from an atomic model by first calculating a real space electron density map for the atoms in vacuo, the excluded solvent, and the hydration shell, and then performs a Fourier transform and spherical averaging of intensities to generate the 1D SAXS profile. PDB2SAS allows you to fit the excluded solvent and hydration shell to best match experimental data.

If you use RAW to run PDB2SAS, in addition to citing the RAW paper, please cite the paper given here.

The written version of the tutorial follows.

1. The easiest way to generate a theoretical profile in RAW using PDB2SAS is simply to plot a .pdb file. In the Files Control Panel go to the theory_data folder, select the 1XIB_4mer.pdb and click the plot button.

   • Note: .cif files are not currently supported in PDB2SAS.

   • Note: PDB2SAS is the default calculator in RAW. However, it is possible that it has been changed to CRYSOL by a user. If so, you can reset the default calculator to PDB2MRC by going to Options->Advanced Options->General Settings and selecting PDB2SAS in the “Default structure calculator” drop down menu.

2. After the calculation is done, RAW will plot the theoretical profile from PDB2SAS on the profiles plot. You should see one profile, 1XIB_4mer.dat

   

3. You can also generate a theoretical profile using the PDB2SAS window in RAW. This gives you more control over the parameters used to generate the profile, using the plot button simply uses the default parameters. Select the “Tools->DENSS->PDB2SAS” menu option to open the PDB2SAS window.

   

4. In the PDB2SAS window, in the models section use the Add button to add the 1XIB_4mer.pdb file to the list of models to calculate a theoretical profile from.

   

5. Click the “Start” button to calculate the theoretical scattering profile for the model.

   • Note: The Status will change to “Running calculation” while running and then to “Finished calculation” when finished.

   

6. After the calculation finishes, the theoretical profile will display in the plot on the right side of the window, and parameters from the profile will be shown in the list at the bottom right of the window.

   • Note: Not all parameters will display. The Data and Chi^2 fields are only calculated if the model is fit to data.

   

7. The PDB2SAS window can also be used to fit the model against experimental data. Go back to the Files Control Panel and load in the glucose_isomerase.dat data. Return to the PDB2SAS window. In the Experimental data section use the Add button, check the glucose_isomerase.dat item in the dialog that appears, and then click OK to add the glucose_isomerase.dat experimental data to the list of data you can fit a model against.

   • Note: Only data that is loaded into RAW can be added to the PDB2SAS experimental data list.

   

8. Click the Start button to calculate the theoretical scattering profile and fit it to the data. After the calculation finishes you’ll see both the theoretical profile, the data, and the uncertainty normalized residual plotted in the right plot, as well as the parameters from the theoretical profile in the list at the bottom right.

   • Note: This will now have all parameters, including the Data and Chi^2 fields that were missing when you calculated a theoretical profile without fitting.

   

9. Click the OK button to close the PDB2SAS window and send the calculated theoretical scattering profile to the Profiles plot. It will appear as 1XIB_4mer_glucose_isomerase_FIT in the Profiles Control Panel.

10. You can also load more than one model and/or profile into the PDB2SAS window to calculate multiple profiles at once. Load the polymerase.dat experimental data into RAW. Select both the polymerase and GI profiles in the Profiles Control Panel, right click on the polymerase.dat profile and select “Other Analysis->Fit Model (DENSS PDB2SAS)”. This will open the PDB2SAS window with the selected profiles already loaded in the Experimental Data section.

    

11. Add the 1XIB_4mer.pdb (GI) and 2pol.pdb (polymerase) models to the PDB2MRC model section.

12. Uncheck the 2pol.pdb model in the Models list. Only items that are checked are used for calculation, so this will let you fit just the 1XIB_4mer.pdb model.

    

13. Click the Start button to fit the 1XIB_4mer.cif model against both experimental profiles. After the calculation finishes you’ll see both experimental profiles and the theoretical profile fit to both measured profiles.

    • Question: Which dataset does the model fit better?

    • Try: Turn off the 1XIB_4mer model and turn on the 2pol model and see how that fits both profiles.

    

14. You can also fit multiple models against a single profile. Check both the 1XIB_4mer.pdb and 2pol.pdb entries in the models list and uncheck the glucose_isomerase.dat experimental data.

    

15. Click the Start button to fit both models against the polymerase data.

    • Question: Which model fits the profile better?

    • Try: Turn off the polymerase profile and turn on the GI profile and see how each model fits that data.

    • Tip: You can also calculate the theoretical scattering profile from multiple models without fitting against data. To do this, uncheck all the data items and calculate the ‘minimal’ theoretical profiles.

    

16. You can fit multiple models against multiple profiles. Check both models and both experimental profiles. Click the start button to fit both models against both experimental profiles.

17. You can export the values in the results table to a csv file. Right click on the table and select Export Data. Save the .csv file in the theory_data folder.

    • Try: Open the .csv file in Excel or another spreadsheet program.

    

18. Click OK to close the PDB2SAS window and send all the fits to the Profiles plot and control panel.

19. You can also adjust the settings for running PDB2SAS from the PDB2SAS window. We’ll do that using some example data. Load the SASDP43.dat experimental data into RAW.

    • Note: This dataset is from the SASDP43 entry in the SASBDB

20. Open a PDB2SAS window and add the Brpt55_M_Zn.pdb model and the SASDP43.dat data.

21. Run the PDB2SAS fitting with the default settings.

    • Note: Notice that the maximum q value for the theoretical fit does not extend all the way to the experimental maximum. This is because the default number of samples (128) is too small. This protein is elongated causing the real space voxel size to be quite large, which results in the maximum q value to be too small.

22. Open a second PDB2MRC window and add the same model and data to it. Set the N samples (real space) option to 256. Run the PDB2SAS fitting with these updated settings.

    

23. You should see that the result using 256 samples extends to the full q range of the experimental data.

    • Note: Setting the N samples to a power of 2 (64, 128, 256, etc.) is best for calculation speed, though any even number can be used.

    • Note: You can explicitly set the voxel size in the Advanced Settings, which will override N samples.

    

24. There are also a number of advanced settings you can set. Expand the Advanced Setting section and (if necessary) scroll down to see the different options. The settings are explained in detail in the PDB2SAS manual.

    • Note: Common settings to change are the solvent density and hydration shell contrast (requires turning off the “Fit solvent” and “Fit hydration shell” options).

    • Tip: If you need all the PDB2SAS outputs (such as the .dat and .fit files) you can check the “Save all outputs to folder” option and provide a folder to save to by clicking the “…” button after the “Save to” field.

    

25. Close the PDB2SAS windows with the OK button to save the fit results to the Profiles plot and control panel.

26. You can save information from a theoretical profile generated by PDB2SAS in RAW as part of a pdf report. Right click on the 1XIB_4mer_glucose_isomerase_FIT item in the Profiles control panel and select “Save report”. In the window that opens click “Save Report” and save the pdf report. If you open the report you will see the usually summary pots and a table with a summary of theoretical profile parameters.