Difference between revisions of "Combined MR-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: lyso2001_scala1.mtz
Lactalbumin model: 1fkq_prot.pdb
Sequence file: hewl.pir

This tutorial illustrates a common molecular replacement/experimental phasing scenario, when refinement is hindered by very strong model bias, but there is some experimental phasing signal available.

Goat α-lactalbumin is 45% identical to hen egg-white lysozyme. Although it is possible to solve lysozyme using α-lactalbumin as a model, it is very difficult to refine the structure, partly because of model bias. Unfortunately, low solvent content of this crystal form limits the ability of density modification to remove the bias. However, one can use anomalous scattering from intrinsic sulfur atoms to improve phases dramatically. It is noteworthy that the anomalous signal from the sulfur atoms is not sufficient for ab initio phasing (it is not possible to locate the anomalous scatterers from the data alone).

  1. Solve the structure with the α-lactalbumin model. Follow the "Molecular replacement tutorial" if necessary.
  2. For a fairer comparison of phase quality, we will treat the molecular replacement solution as a source of experimental phase information. (If you use the "automated model building starting from PDB file" mode, the current version of ARP/wARP will be able to build the structure, but older versions coupled with older versions of Refmac5 failed.) Do a quick solvent flattening with DM using AUTO cycles (input Hendrickson-Lattman coefficients, and set PHIO=PHIB and FOMO=FOM). The best results are obtained if DM starts with the map produced by Phaser instead of a simple FOM-weighted map. In CCP4 6.1, you can check the box labeled "Input starting map coefficients" and enter FDM=FWT and PHIDM=PHWT. For older versions of CCP4, you have to start the job with "Run&View Com File", edit the LABIN keyword to include the following: FDM=FWT PHIDM=PHWT, and then run the job.
  3. Start up ARP/wARP Expert System in "automated model building starting from experimental phases" mode. Start from the DM phases, and include HL coefficients for phase restraints (use the ones from Phaser).
  4. Now add the S-SAD phase information. Bring up the GUI for Phaser in the Experimental Phasing module
  5. All the yellow boxes need to be filled in.
    • Set "Mode for experimental phasing" to SAD with molecular replacement partial structure.
    • Set "LLG-map calculation atom type" to S.
    • Under the "Define atoms" heading, set "Partial structure" to the molecular replacement solution (output PDB-file) you have obtained in step 1.
  6. Run Phaser after you entered all the information.
  7. Solvent flatten with DM using the same protocol as in step 2.
  8. Run ARP/wARP Expert System using the same protocol as in step 3.
  9. How many anomalous scatterers has Phaser found? Check them against the model and guess what they may be! Why is it not important to specify the exact element type in this case?
  10. If you did not know the correct space group, would you have to run Phaser twice?
  11. Compare the two ARP/wARP runs! Which one has built more residues?