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The images have been archived in two parts.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 &

(C) Analysis

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

firefox report-grade-LIG/index.html

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.