SAD using CCP4i

From Phaserwiki
Revision as of 16:03, 10 April 2013 by Randy (talk | contribs) (Update to describe Phaser pipeline instead of old standalone Phaser-EP GUI)

Data for this tutorial are found here

Reflection data: iod_scala-unique.mtz
Heavy atom sites: iod_hyss_consensus_model.pdb
Sequence file: hewl.pir

This tutorial demonstrates SAD phasing using the Phaser pipeline.


Lysozyme will readily crystallise from a wide range of conditions and yield several crystal forms. One of those, tetragonal lysozyme, is particularly well suited for halide soaking, since it grows from a high concentration of sodium chloride. A dataset has been collected from a lysozyme crystal soaked in 0.5 M potassium iodide. A strong anomalous signal has been detected, and locations of anomalous scatterers have been found.

  1. Start the ccp4 GUI by typing ccp4i at the command line.
  2. Make a new project called "phaser_tute" using the Directories&ProjectDir button on the RHS of the GUI. Set the "Project" to "phaser_tute" and "uses directory" to the directory where the files for this tutorial are located, and make this the "Project for this session of the CCP4Interface". You will then be able to go directly to this directory in the GUI using the pull-down menu that appears before every file selection.
  3. Go to the Experimental Phasing module, in the yellow pull-down on the LHS of the GUI
  4. Open the Automated Search & Phasing tab, then bring up the GUI for the Phaser SAD pipeline
  5. You can choose (near the top) whether to use SHELXD (if you have the SHELX package installed) or HYSS (if you have the Phenix package installed) to find the substructure before running Phaser. If not, you can use a predetermined substructure, such as the PDB file included with this tutorial.
  6. You can also choose whether or not to run Parrot to carry out density modification and Buccaneer to carry out a very preliminary model building, in the pipeline after Phaser.
  7. All the yellow boxes need to be filled in.
    • Make sure that "Mode for experimental phasing" is set to Single-wavelength anomalous dispersion (SAD). This is the default.
    • Note that Phaser requires F(+), SIGF(+), F(-) and SIGF(-) and not the FP and DANO used by some other programs.
    • Do not forget to change the "LLG-map calculation atom type" from the default Se to I.
    • In a general case one should set "Enantiomorph choice" to Both enantiomorphs. Why is this not necessary in this case?
    • It is also a good idea to fill in the TITLE.
  8. When you have entered all the information, run Phaser.
  9. Look at the "Final phasing statistics" table at the end of the logfile.
    • There is some explanation on important logfile sections in the Phaser Wiki
  10. Look through the log file and identify the workflow. How many cycles did Phaser need to reach convergence? What are the convergence criteria?
  11. If you ran the whole pipeline, look at whether one hand gave better statistics than the other in the Parrot and Buccaneer steps. Look at the better Buccaneer model in the corresponding Parrot map, using coot.