Tutorial: Experimental phasing with Phaser in the ccp4i interface

All files for this tutorial are distributed from the Phaser web page http://www-structmed.cimr.cam.ac.uk/phaser/tutorial/phaser-ep-tutorial.tar.gz.

Tutorial 1: SAD

SAD phasing starting from heavy atom positions using Phaser.

• Reflection data: iod_scala-unique.mtz
• Heavy atom sites: iod_hyss_consensus_model.pdb
• Sequence files: hewl.seq & hewl.pir (formatted for ARP/wARP)

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 (using the program HySS from the Phenix package–SHELXD could equally well have been used).

1. Start the ccp4 GUI by typing ccp4i at the command line.
2. Make a new project called "phaser_eptute" using the Directories&ProjectDir button on the RHS of the GUI. Set the "Project" to "phaser_eptute" 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 by choosing Phasing&Refinement->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.
• The default wavelength, CuKa, is correct in this case.
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?
• Use the Phaser documentation: http://www-structmed.cimr.cam.ac.uk/phaser/documentation/phaser-2.1_key.html
9. Look at the map produced by Phaser. The correct map coefficients, FWT/PHWT, will be chosen as the default by coot.
• Run DM to improve the map by solvent flattening, 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. You can get the solvent content from the Phaser log file. Look at this map in coot (choosing map coefficients FDM/PHIDM), turning on the map skeleton to see how easily it could be traced.
• If you like, run ARP/wARP to build a model. The best results are obtained if you refine with the MLHL target in the Refmac5 part of ARP/wARP. Choose the Hendrickson-Lattman coefficients from Phaser (HLA/HLB/HLC/HLD), because the ones produced by DM (HLADM/HLBDM/HLCDM/HLDDM) tend to be overly optimistic. You can save time by reducing the default number of build cycles (about 5 should be enough).

Tutorial 2: MR+SAD

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.

• Reflection data: lyso2001_scala1.mtz
• Lactalbumin model: 1fkq_prot.pdb
• Sequence files: hewl.seq & hewl.pir

Goat α-lactalbumin is 40% 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, as described above.
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). Reduce the number of Build Cycles to save time.
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. Look at the maps before and after solvent flattening. Compare with the maps generated with just the molecular replacement model phases.
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?