CCP4i: Graphical User Interface
	Tutorial 6

Refinement

Run Refmac

Please note that this tutorial refers to refinement with Refmac before version5.0. This can only be run through the Program List. A tutorial for refinement with Refmac5 (now the preferred option) is available elsewhere.

Everything is now in place to run Refmac: coordinate file, reflection file and restraints description.

Refmac - a simple example

Select Run Refmac
on the left, in the Refinement module (you may have to select this module first). This opens the Task Window for Refmac.

The first four lines below the gold-coloured bar constitute the Protocol folder. The options in this folder represent the key decisions which may change the defaults and options in the rest of the Task Window.
Enter a 'Job title' that means something to you. Try to avoid special characters (like $ or !).

To run Refmac completely in default modes, you only have to fill in the Files folder (i.e. the next five lines, concerned with input-and-output reflection and coordinate files) and the "Setup Protin" folder, and set Refmac running.

1. In the 'MTZ in' line, select rnase_exam or ccp4itut
   from the directories pop-up menu,
   and Browse for rnase18.mtz.
   The Interface automatically selects the columns for the native structure (FP and SIGFP) from the reflection file.
   In case you are using the output MTZ file from Tutorial 1 - Preparing data, you should Browse on the directory ccp4itut for the appropriate MTZ file (if you have run UNIQUEIFY more than once, you will also need to use the correct serial number).
2. The Interface automatically assigns a name and path for the output reflection file. In a 'real' project, you may want to keep this file permanently, which is what the Interface suggests at this point. For the purpose of this tutorial, it is probably better to put it on the TEMPORARY directory, from which it will be deleted automatically later. So
   in the 'MTZ out' line, select TEMPORARY
   from the directories pop-up menu. The Interface keeps the name, but changes the directory path.
3. In the 'PDB in' line, select rnase_exam
   from the directories pop-up menu,
   and Browse for rnase.pdb.
4. In the 'PDB out' line, select TEMPORARY
   from the directories pop-up menu, so that the output coordinate file may be deleted automatically later, and will not take up disk-space permanently (see above).
5. In the folder "Setup Protin", click on the pop-up menu and select from selected Protin def file.
   The def file you should use is on $CCP4I_top/test_data, and is called protin-ccp4itut.def.
6. Select Run > Run Now
   at the bottom left of the Task Window to set Refmac running.

In the Main Window of the Interface, you will notice information appearing in the Job list in the middle of the window: a job number, the time the job started running, the status of the job (STARTING, RUNNING, FINISHED), the type of job (refmac) and the job title.

Refmac - evaluating the input and output

See also Tutorial 1 - Looking at files.

Select View Command Scripts
from the View Files from Job pop-up menu. In the FileViewer, the command scripts for Protin and Refmac are shown. Protin is described in Tutorial 6 - Protin. The Refmac command script contains the following:

symmetry

P212121 - extracted from the MTZ file

refinement

type - restrained

residual - maximum likelihood

method - conjugate direction

B-factors - isotropic

ncyc

5 cycles of Refmac are run before Protin is run again; only 1 'macrocycle' of Refmac+Protin is run

bins

the data is split into 20 resolution bins

scaling

resolution range - all data to 1.83

type - bulk solvent scaling

LSSC (least squares scaling)

flag to indicate the following scaling subkeywords apply to estimation of scale between Fo and Fc:

anisotropic - anisotropic scaling of Fo and Fc; the program default is NOT to apply anisotropic scaling, but since it is appropriate in most cases, the Interface default is different.

ncycles - 10 cycles

weighting of X-ray and geometry in the residual

matrix - weighting using average diagonal terms of X-ray and geometry Hessians; value of 0.5 ensures X-ray has half the weight of geometry

monitoring

few - only overall statistics will be monitored

input labels

output labels

end

Must be last keyword. "Time to do work."

Select View Log File
The log file is shown in a FileViewer window. It can be searched by clicking the Find button and typing a string (e.g. 'overall_R_factor'), and then clicking the Highlight button to activate the search. There are 7 hits for this string - 1 before refinement, 1 after every cycle of refinement, and 1 more after the structure factor calculation (this last one is a feature of the CDIR refinement method).
Another quick route to the R-factor information, is the Show Summary pop-up menu.
Select Default summary
(the Select summary definition file does not work very well at the moment). The FileViewer window now displays CCP4i- and CIF-specific lines, taken from the full log file. With the big black arrows above and below the scroll bar on the right, you can scroll a whole window at a time. The Find tools work as above. The R_factors change from 0.238 (both the conventional and free R factors) to 0.195 and 0.228 (conventional and free R factor, respectively) in this run of Refmac.
Quit
the FileViewer.

Select View Log Graphs
The Loggraph window displays a list of the Tables that appear in the log file: R-factor analysis and Figures-of-merit for every cycle of refinement, and an extra R-factor analysis concerning the structure factor calculation (see above). The R-factor analysis tables contain two graphs each.
Select the first Rfactor analysis table and the graph <Rfactor>
Viewing all subsequent Rfactor analysis/Rfactor graphs gives a nice overview of the course of this refinement. Note that the scale of the y-axis changes as the highest Rfactor dives below 50%.
Select the first Fom table
The corresponding graph is opened automatically. Viewing all subsequent <Fom> graphs shows that Refmac works towards a flat distribution.
Exit
The Loggraph Utility.

The Input files .. and Output files .. for this task are the input and output reflection files (which can be viewed with the FileViewer once the Interface has run MTZDMP on it) and the input and output coordinate files (which are shown in the FileViewer directly). All can be searched with the Find tools as before. In addition, for the MTZ files there is an option to List More Info. Through this, you can acquire more information from MTZDUMP (most of the program's normal options available) or from SFTOOLS (mainly to list specific reflections).
Refmac does not allow for the 'history' of the MTZ file to be included in the output MTZ file, so this information will be lost. Furthermore, somewhere along the line the low resolution limit is interpreted slightly differently, and one reflection is lost from the file (as it stands at the moment; but this may be fixed somehow. Watch this space).

More information

For more information on the Refmac program, see REFMAC.

The Interface documentation on features from this tutorial can be found at: