MR using CCP4i:TOXD
Data for this tutorial are found here
Reflection data: toxd.mtz Structure files: 1BIK.pdb, 1D0D_B.pdb Sequence file: toxd.seq Sequence alignment: toxd_sequences.aln
This tutorial demonstrates the procedure to make and use ensemble models in Phaser. An ensemble model is a set of alternative models superimposed on each other so that Phaser can make a statistically-weighted ensemble average model from them. The ensembling procedure is contrasted with another approach, which is to use the individual models as separate alternatives to search for the same component. Generally, if the alternative models superimpose well on each other, it is better to use the ensembling approach. However, if the alternative models represent different conformations, it may be better to test them separately.
α-Dendrotoxin (TOXD, 7139Da) is a small neurotoxin from green mamba venom. You have two models for the structure. One is in the file 1BIK.pdb, which contains the protein chain from PDB entry 1BIK, and the other is in the file 1D0D_B.pdb, which contains chain B from PDB entry 1D0D. 1BIK is the structure of Bikunin, a serine protease inhibitor from the human inter-α-inhibitor complex, with sequence identity 37.7% to TOXD. 1DOD is the complex between tick anticoagulant protein (chain A) and bovine pancreatic trypsin inhibitor (BPTI, chain B). BPTI has a sequence identity of 36.4% to TOXD. Remember that models making up an ensemble must be superimposed on each other, which has not yet been done with these two structures. At relatively low sequence identity, it is also a good idea to trim unconserved loops and the ends of non-identical side chains, so that the model comprises a conserved core.
- Start the ccp4 GUI by typing ccp4i at the command line.
- 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.
- Go to the Molecular Replacement module, in the yellow pull-down on the LHS of the GUI
- Under Model Generation within the Molecular Replacement module, bring up the GUI for Sculptor
- Run sculptor on each of the MR models, using the alignment information in toxd_sequences.aln. Just use protocol 12 (the overall best choice found in a series of trials) of the Predefined protocols to modify the starting models.
- It is a good idea to fill in the TITLE.
- Under Model Generation, now bring up the GUI for Ensembler
- Run ensembler to combine the two sculpted models. It is more convenient to put them together into a single PDB file as a merged ensemble, but it is also possible to provide separate PDB files to Phaser when defining an ensemble. By default, sculptor has changed the sequences of the two models to match the target sequence, so it is not necessary (or appropriate) to provide the sequence alignment file here.
- Check the resulting merged ensemble PDB file using a graphics program such as coot.
- Bring up the GUI for Phaser
- All the yellow boxes need to be filled in.
- It is a good idea to change the Ensemble id from the default.
- By default, Phaser will attempt to use intensities rather than amplitudes. Because there are only amplitudes available for this data set, uncheck the box labelled "Input data are merged intensities".
- The merged ensemble file from ensembler contains REMARK records containing the sequence identities (placed there by the sculptor step), so you can choose "from pdb REMARK" in the pulldown for "Similarity of PDB..."
- Don't forget to enter a TITLE.
- When you have entered all the information, run Phaser.
- Has Phaser solved the structure? What was the LLG of the best solution? What was the Z‑score of the best translation function solution?
- The meaning of the Z‑score is given in the documentation
- Look though the log file and identify the anisotropy correction, rotation function, translation function, packing, and refinement modes. Draw a flow diagram of the search strategy.
- How many potential solutions did Phaser find or reject at each stage? What were the selection criteria for carrying potential solutions forward to the next step in the rotation and translation functions? How many other selection criteria could have been used, and what are they?
- Use the documentation
- Run Phaser again without using ensembling i.e. run a job testing 1BIK and 1D0D separately as alternative models. This is done in the ccp4i GUI by opening the "Additional Search parameters" pane, choosing "on" for "Allow search with alternative ensembles" (which reconfigures the "Search parameters" pane) and then entering the alternative models.
- What are the LLGs of the final solutions? What are the Z‑scores of the translation functions? Was ensembling a good idea? You could also run Phaser on the models before sculpting, to see if this has improved their quality.