Examples of running Phaser in CCP4i

May 2006

These examples are based on the keyworded tutorials from the Phaser website. There are now similar tutorials available at http://www-structmed.cimr.cam.ac.uk/phaser/faq.html. The examples were run using Phaser 1.3.2 from CCP4 6.0.

The Phaser documentation is also helpful: http://www.ccp4.ac.uk/dist/html/phaser.html

Example 1: TOXD with a simple ensemble

  1. Start the Phaser interface within CCP4i
  2. Enter the title Example 1 toxd fast rotation search
  3. Select Mode for molecular replacement to be rotation search using fast rotation function
  4. Select the input MTZ file to be toxd.mtz - the labels should automatically default to FTOXD3 and SIGFTOXD3
  5. Select the spacegroup to be that of the MTZ i.e. P212121
  6. Set up the details for component #1 in the Composition of the asymmetric unit folder: This gives Phaser an idea of what it is looking for.
  7. Specify the search model to use for the rotation function in the folder Define ensembles. An ensemble is Phaser's name for a search model, which can be made up of multiple coordinate files - to start with with we will use just one model in our ensemble: This information gives Phaser an idea of how similar the search model is to the target - alternatively, the expected RMS coordinate error can be used instead.
  8. In the Search details folder tell Phaser to Perform search using toxd i.e. the search model that you just defined.
  9. Click on Run

What to look for in the output

The Z-score for each solution is used to get an idea of how likely that solution is to be the true solution. The section How to know whether Phaser has solved it in the Phaser documentation gives an indication of appropriate Z-score values for rotation and translation functions.

(The Z-score is the number of standard deviations that the solution peak height is above the average peak height.)

This job should create three output files:

Example 2: extending the ensemble

We can improve our search ensemble from example 1 by extending it to include more information in the form of additional coordinate files. Phaser will take all the models within the ensemble and merge them into an averaged model. The idea is that parts of the model which are similar will be emphasised while dissimilar parts will be weighted down.

In order for Phaser to be able to combine models within an ensemble it is necessary that the PDB files are properly superimposed. Within CCP4i you can use the Superpose Molecules task (in the Coordinate Utilities module) to do this. It is recommended that you also check the superposition using a graphics program.

  1. In the Phaser task window, set the title to Example 2 toxd fast rotation search with extended ensemble
  2. In the Define ensembles folder add a second PDB file
  3. Run the task

Note that if the coordinates are not superimposed (for example if you use the file 1BIK.pdb rather than the superimposed coordinates 1BIK_2_1D0D_B.pdb) then Phaser will probably stop with a fatal error.

At the end of this run you should get a single rotation search solution with a better Z-score than any of the solutions from the previous example using just a single model in the ensemble.

Example 3: TOXD translation search

Having obtained a rotation function solution in example 2, we can now use this to perform a translation search to get a complete solution.

  1. In the Phaser task window, set the title to Example 3 toxd fast translation search with extended ensemble
  2. At the top of the window, set the Mode for molecular replacement to translation search using fast translation function
  3. In the Search details folder (below the ensemble definitions) select the .rlist file from the previous run - this means that the solutions from that run will be used in the translation search.
  4. Run the task

Looking at the .sol file indicates a translation function Z-score of 9.4 - since this is higher than the value of 8 recommended by the documentation, it is likely that this is the correct solution.

To see the difference that using the two model ensemble makes, try running the translation search with the simple ensemble from example 1. There are more solutions however Phaser still picks out a solution with a Z-score of 9.4.

Example 4: Beta-Blip using auto search

This last example is a more complex example which uses Phaser in its automated search mode. This mode performs rotation and translation function steps followed by a packing step to check for clashing solutions.

BETA/BLIP is the beta-lactamase/beta-lactimase-inhibitor complex. The BETA component constitutes 62% of the scattering material and is relatively easy to locate, whereas the BLIP component is much harder to locate. However once Phaser has located the solution for BETA, it is able to use this information to find BLIP.

  1. Start the Phaser interface
  2. Enter the title Example 4 Find BETA and BLIP using auto search
  3. Select Mode for molecular replacement to be automated search (this should be the default)
  4. Select the input MTZ file to be beta-blip.mtz - the labels should automatically default to Fobs and Sigma
  5. In the Define data folder, select Run Phaser with the mtz space group and enantiomorph (if applicable). This is because although the spacegroup recorded on the mtz file is P3221, in this case the other hand is also a possibility.
  6. Set up the details for the BETA and BLIP components in the Composition of the asymmetric unit folder:
  7. Specify the ensembles in the folder Define ensembles. There will be one ensemble for each of the components.
  8. In the Search details folder, set up the searches to be performed.
  9. Optionally: in the Additional parameters folder, check the box for Permute search set, and select on. This means that both search orders (BETA first, BLIP second and BLIP first, BETA second) are tried. (In practice this option is not necessary for this case, as the larger molecule should be easier to find first.)
  10. Click on Run

Note that it can take some time to run this example, and that the resulting log file can be very long.

At the end there are a number of output files - as well as .sol, .rlist, and .pdb files there should also be:

Beyond the examples

Phaser supports a large number of difference options and protocols. Some options that might be of interest include:

Different ways to specify composition of the asymmetric unit

In the above examples the composition was specified in terms of the molecular weight of each of the components. However it is also possible to use:

Different ways to specify models in ensembles

Coordinate files are common ways to specify molecular replacement models. However Phaser allows specification of models in other ways: