Parent directory: 9_easy
Sequence file: 4hg7.fasta
Reflection data: 4hg7.mtz
Structure files: 1t4e.pdb

1. Introduction

This is an easy but not completely straightforward MR example. Illustrates importance of model editing.

Assume that 4hg7.mtz presents your experimental data. Try to solve and refine this structure starting from 1t4e.pdb and using any tools you like.

The asymmetric unit of the template structure 1t4e contains two molecules of a fragment of a human oncoprotein MDM. The target structure 4hg7 contains a complex of an almost (but not completely!) identical fragment of the same protein with the ligand.

Fragments are very similar, same initial sequence, model 16-111 (+ 5 residues of expression tag, invisible in the structure), target 17-108. So at a first glance there is no need to modify the model.

The ligand is as follows:

A clear density for this ligand will indicate that the structure is solved. At this point you may also exercise with Coot > Calculate > Ligand Builder to generate description of this ligand and to refine the complex.

2. Instructions

(1) Find the files listed above in the tutorial directory.

(2) How many molecules are there in the asymmetric unit of the target structure? To answer this question, use files 4hg7.seq and 4hg7.mtz and Cell Content Analysis task in CCP4I (CCP4I > Molecular Replacement > Analysis > Cell Content Analysis).

(3) Extract chain A from the file 1t4e.pdb into 1t4e_A.pdb. (CCP4I > Molecular Replacement > Edit PDB Model File. Change selections in the second line of the task menu to "pdbset" and "perform selection for output PDB file". Other selections should be clear)

(4) Run molrep (don't use sequence- assuming it's the same protein, and almost same length constructs) and examine the table at the end of the log-file. Is the molrep solution convincing? Try refinement. Did it go smoothly and produce interpretable density? Do you see the ligand density?

(5) Run phaser. Did you notice any unusual message? Refine the model. Do you think phaser found a solution?

(6) Ways to proceed: Phaser: increase phaser's tolerance to clashes, or remove three C-terminal residues from the model.

Molrep - use sequence now. Or remove three C-terminal residues.

(7) Compare solution from (4) or (5) with the solution from (6) using Csymmatch (CCP4I > Program List > csymmatch; define input files and tick the box "Apply origin correction and hand correction).

(8) Overlap the structures in Coot. See the shift between the right and wrong solution. Go to Draw>cell and symmetry>show symmetry>symmetry by molecule-display as Calpha s - and see why 3 residues made such a big difference.

This example shows how a small modification of the model can make a big difference.

*) like cloning in a way, when two-three truncated residues could make the construct soluble.