Difference between revisions of "SAD using CCP4i"

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Revision as of 10:41, 9 July 2009

All files for this tutorial are distributed from the Phaser web page

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

This tutorial demonstrates SAD phasing starting from heavy atom positions using Phaser.

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. Bring up the GUI for Phaser
  5. 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.
  6. When you have entered all the information, run Phaser.
  7. 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
  8. Look through the log file and identify the workflow. How many cycles did Phaser need to reach convergence? What are the convergence criteria?