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SAD and MR-SAD phasing with the Phaser GUI

Input files and mandatory parameters
Output
Frequently Asked Questions
References

Since the two Phaser GUIs use a common base, much of the experimental phasing interface is similar to the Phaser-MR GUI. This section only covers features unique to SAD phasing, including instructions for MR-SAD.

Phaser is used internally in the AutoSol wizard, which automates a complete experimental phasing pipeline including heavy atom search, phasing, density modification, and preliminary model-building. (It is also capable of running MR-SAD experiments.) For that reason, we recommend trying AutoSol first, and falling back to standalone Phaser for more difficult problems where finer control over parameters is required.

This document only covers configuration of the Phaser GUI; for a detailed explanation of the various terms and search procedures referenced here as well as interpretation of results, consult the documentation for AutoSol or Phaser, or the Phaser WIKI.

Input files and mandatory parameters

Phaser requires a reflections file (any format) containing anomalous data, and a file containing heavy atom sites, in either PDB or SHELX format. Either the wavelength or the energy used is also required. To run an MR-SAD experiment, change the mode to "MR-SAD phasing", and enter a partial model (PDB file) and the expected variance from the actual structure (RMSD or percent identity). If you are using normal SAD mode, you may choose whether to use the given enantiomer ("hand") of the heavy-atom sites or the inverse or both; in MR-SAD, the chirality of the partial model restricts the sites to the given enantiomer.

images/phaser_sad_main.png
The input for composition is identical to the Phaser-MR GUI. Heavy atoms may be either standard elements from the periodic table, or cluster compounds such as tantalum bromide or tungsten clusters.

Output

Depending on the choice of hand, Phaser will output one or two sets of files containing the heavy atom sites (PDB format) and phased reflections (MTZ), including map coefficients and Hendrickson-Lattman phase distributions (useful for refining with experimental phase restraints). This map is usually very noisy and difficult to interpret, and typically requires density modification to become useful.

Frequently Asked Questions

  • How do I tell Phaser to look for more anomalous scatterers? Click on the "Composition and sites" tab. A control at the bottom labeled "Enable substructure completion by log-likelihood gradient maps" turns on this feature (and is already on by default). However, Phaser also needs to be told what type of scatterers to look for. Clicking the "Options..." button opens a new window which will allow you to enter scatterer symbols (typically element codes). You may enter as many of these as you think are present and ordered in your crystal - note however that at the wavelengths commonly used for macromolecular crystallography, some atom types may be difficult to distinguish (such as sulfur and chlorine).
  • How do I phase a structure with tantalum bromide clusters in Phenix? This is possible, but not really optimal at present. Phaser has built-in support for Ta6Br12 clusters, which have the scatterer type "TX". However, they are represented as point scatterers rather than the actual cluster. Therefore, although Phaser can perform LLG substructure completion, it will output sites as single TX pseudo-atoms, rather than the actual cluster.
images/phaser_ep_llg.png

References