This tutorial originally comes from the Phaser
The files for this tutorial can be found at:
MR using TOXD
This tutorial demonstrates the ensembling procedure in Phaser.
α-Dendrotoxin (TOXD, 7139Da) is a small neurotoxin from green mamba
venom. You have two models for the structure. One is in the file
1BIK.pdb, which contains the protein chain from PDB entry 1BIK, and
the other is in the file 1D0D_B.pdb, which contains chain B from PDB
entry 1D0D. 1BIK is the structure of Bikunin, a serine protease
inhibitor from the human inter-α-inhibitor complex, with sequence
identity 37.7% to TOXD. 1D0D is the complex between tick
anticoagulant protein (chain A) and bovine pancreatic trypsin
inhibitor (BPTI, chain B). BPTI has a sequence identity of 36.4% to
TOXD. Note that models making up an ensemble must be superimposed on
each other, which has not yet been done with these two structures.
- Use the SSM superpose option in coot to superimpose 1BIK on
1D0D_B, saving the resulting coordinates in 1BIK_on_1D0D.pdb.
Go to the Molecular Replacement module, in the yellow
pull-down on the LHS of the GUI
Bring up the GUI for Phaser
All the yellow boxes need to be filled in. Note that in this
example, the ensemble is the actual ensemble containing
superposed structures. In addition:
- Alternatively, superpose molecules using Gesamt (Molecular
Replacement > Utilities > Superpose Coordinates). This
may be the best option if Coot is slow in the workshop setup.
When you have entered all the information, run Phaser.
Has Phaser solved the structure? What was the LLG of the best
solution? What was the Z-score of the best translation function
- it is a good idea to change the Ensemble id from the
- it is also a good idea to fill in the TITLE.
Look though the log file and identify the anisotropy
correction, rotation function, translation function, packing,
and refinement modes. Draw a flow diagram of the search
How many potential solutions did Phaser find or reject at
each stage? What were the selection criteria for carrying
potential solutions forward to the next step in the rotation and
translation functions? How many other selection criteria could
have been used, and what are they?
Run Phaser again without using ensembling i.e. run two jobs,
one using 1BIK only and the other using 1D0D only as models.
What are the LLGs of the final solutions? What are the Z-scores
of the translation functions? Was ensembling a good idea?
- The meaning of the Z-score is given in the documentation