Pipedream Tutorial 4

This example illustrates the use of Pipedream where the input are the unprocessed x-ray images, rather than a pre-processed data set.

The example used is a lysozyme dataset, which has been complexed with HEPES.

The images are freely available for download at http://www.helmholtz-berlin.de/forschung/oe/em/soft-matter/forschung/bessy-mx/tutorial/experiment-5_en.html.

The images have been archived in two parts. Click on both of the links to download the images and then unpack and uncompress them.

The reference structure is PDB id: 1DPW

1dpw.mtz the structure factors as supplied by the pdb for 1dpw, converted to mtz format
1dpw.pdb the model coordinates as supplied by the pdb for 1dpw

(A) Restraint dictionary generation

Generate a restraint dictionary for HEPES with grade, again using a smiles

grade 'OCCN1CCN(CC1)CC[S](O)(=O)=O' -resname LIG

(B) Pipedream run

The reference structure 1DPW contains some small molecules that need to be removed prior to use:

egrep -v " TRS | MRD " 1dpw.pdb > input.pdb

We are now ready to run Pipedream as follows:

pipedream -imagedir exp5/data -xyzin input.pdb -hklref 1dpw.mtz -rhofit grade-LIG.cif -postref -rhothorough -nofreeref -d pipe &
  • Rather than use pre-processed data, as in the previous examples, we are now using the unprocessed images. The use of -imagedir exp5/data tells Pipedream where the images are located.
    • Note that the use of -imagedir and -hklin are mutually exclusive. You can specify one or the other, but NOT both.
  • The reference mtz file (1dpw.mtz) DOES NOT contain a freeR set and therefore a new set will be automatically calculated. The -nofreeref flag MUST be specified in this case to acknowledge this.

(C) Analysis

The main output file summary.out shows that the job has run to completion and without any errors.

  • The second main section indicates that the input data was an unprocessed set of images and that it has been successfully processed with autoPROC. The salient processing statistics are tabulated. For a more detailed summary of the data processing, look at the autoPROC standard output file process.out.
    • Note that autoPROC has been run having been told to index the data with the cell dimensions and space group read from the reference structure. If autoPROC is unable to index data in the specified cell / space group then Pipedream will terminate with an appropriate message.
  • The rest of the file indicates that the structure has seemingly been well refined, Rhofit has found a good solution and it has been post-refined well.
    • The final statistics (R = 16.4, Rfree = 19.5) are somewhat better than those quoted on the web site listed above.
  • Look at the output from buster-report:
firefox report-grade-LIG/index.html
  • Look at the overall statistics listed on the main tab and also look at the "Ligand report" tab. There is nothing there of any concern. The refinement statistics look very good and the ligand geometry looks good too.
  • Now look at the "Molprobity analysis" tab. For the most part, this looks OK, however the number of residues with "bad angles" is a little high. Some manual inspection and intervention may warranted. Again, Pipedream is NOT designed to give a "highly polished", deposition ready structure.

More importantly, is the ligand solution correct? Pipedream will only select what Rhofit classifies as its "best" solution for post-refinement.

So, lets look directly at the Rhofit output:

visualise-rhofit-coot rhofit-grade-LIG

Coot will open, initially displaying its "best" solution and the difference density map.

In the pop-up window, select the "Protein visible" button to also show the protein and the 2fofc density.

popup.png

Click on the "Next position" tab to scroll through all of the potential solutions that Rhofit built. Solution 0 appears to be correct.

From within Coot, open the final post-refined structure as well and compare it with Solution 0. The post-refined ligand is shown in red in the picture below.

solution0.png

This shows that refinement tweaked the position of the ligand as fit by Rhofit, but confirms the validity of the solution. Note the electrostatic interactions between the two nitrogen atoms of the piperazine ring and an ASP carboxyl oxygen and a water respectively.