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 from released PDB entry 6CBX - "human SET and MYND Domain Containing protein 2 with MTF1497"

The images are available for download at https://data.proteindiffraction.org/sgc/SMYD2_6cbx.tar.bz2

Click on the link to download the images and then unpack and uncompress them.

The reference structure and input model used is from the related PDB structure 6CBY

6cby.pdb the model coordinates as supplied by the pdb for 6CBY

(A) Restraint dictionary generation

Generate a restraint dictionary for ligand MTF1497 with grade, again using a smiles

grade 'Cc1cc2n(cnc2c(N)n1)[C@@H]3CN(C3)C(=O)c4ccn(C[C@H]5CCN(CC5)[C@@H]6CCCCC=C6)c4' -resname LIG

(B) Pipedream run

The reference structure 6CBY contains solvent and a ligand that need to be removed prior to use:

egrep -v " EW4 | HOH " 6cby.pdb > input.pdb

We are now ready to run Pipedream as follows:

pipedream -imagedir <dir> -nofreeref -useaniso -apcommands "ReversePhi=yes" -xyzin input.pdb \ 
-chains "A B" -remediate -sidechainrebuild -rhofit grade-LIG.cif -xclusters 2 -postref -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.

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

rhofit-a.png

Now select the "Next search region" tab to look at the second binding site. Again, solution 0 appears to be correct.

rhofit-b.png

Look at the final post-refined structure and map using visualise-geometry-coot:

visualise-geometry-coot postrefine-grade-LIG

chain A:

postref-a.png

chain B:

postref-b.png