PipedreamTutorial2

Pipedream Tutorial 2

This example, again using beta-secretase structures, illustrates a slightly more complex situation which requires specification of extra commands and changing defaults for pipedream.

For this tutorial, the reference structure is PDB id: 3NSH and the experimental data is taken from PDB id 3I25.

• (A) Restraint dictionary generation
• (B) Pipedream run
• (C) Analysis

(A) Restraint dictionary generation

3i25 was soaked with a large and very flexible inhibitor, ligand code MV7.

• Once again we need to generate a restraint dictionary with Grade, again using a smiles description of the ligand as input:

grade -in MV7.smiles -resname LIG 

(B) Pipedream run

The structure that is being used as the "reference" in this case, 3NSH, is not a native structure but has a bound ligand (ligand code 957) in the active site.

• So before use, the ligand MUST be removed from the reference structure:

grep -v " 957 " 3nsh.pdb > input.pdb

We are now ready to run Pipedream:

pipedream  -hklin 3i25.mtz -xyzin input.pdb -hklref 3nsh.mtz \
-chains "A B C" -rhofit grade-LIG.cif -xclusters 3 -rhothorough \
-postref -d pipe1

• Unlike Tutorial 1, the beta-secretase structures we are using here have crystallised with 3 monomers in the asymmetric unit.
• The -chains keyword affects the limited MR module, and tells phaser to move the 3 protein chains independently (the default is to move the entire structure as a rigid body).
• Pipedream does not automatically determine number of expected binding sites. The -xclusters 3 keyword is specified to tell Rhofit that there are 3 copies of the ligand to be fitted. The default is to only fit the ligand into the single best site.
• The ligand (MV7) is very large (MW ~ 900) and flexible, so we have specified -rhothorough. This instructs Rhofit to run more trials than usual in trying to fit the ligand.

(C) Analysis

The main output file summary.out indicates that the job has run as expected.

The initial refinement has been successful and Rhofit has "successfully" fit the ligand into each of the 3 protein chains. The "best" solution has been post-refined with very good statistics - R = 16.9%, Rfree = 20.0%.

Again, the real test is whether or not the ligand has been correctly fit and refined.

The image below shows the ligand in chains A, B and C.

The orientation of all 3 copies is similar and in each case the ligand fits the density (with little difference density present).

Furthermore, they are a good match to the original 3i25 structure.

Look at the buster-report output:

firefox report/index.html

The ligand analysis is good with no outliers and the molprobity analysis also looks good. Again, there is clearly some room for improvement in the structure - this will need manually correcting in Coot and a subsequent re-refinement.