REFMAC5 Tutorial - Part 1

Simple Refinement

In this tutorial, we will use REFMAC5 to perform simple refinement of a ribonuclease structure.

A basic working knowledge of the CCP4i2 GUI is assumed.

Before We Start

Required files:

rnase.pdb - the current model;
rnase.mtz - the reflection data.

Please click these links to access the files and save them to your computer.

Simple Refinement Using REFMAC5 From Within CCP4i2
Adjusting The Geometry Weight
Inspecting the Electron Density

Simple Refinement Using REFMAC5 From Within CCP4i2

We will begin by performing simple refinement of the ribonuclease structure:

Open the CCP4i2 GUI, and create a new project as appropriate.
Open the REFMAC5 task interface, which can be found in the Refinement module.
Enter a meaningful job title, e.g. "Tutorial Example 1 - Simple Refinement".
Provide the file rnase.pdb in the "Atomic model" field. You can either browse for the file, or drag and drop it into the field.
A window will appear, allowing you to provide a description for the file. Click "OK".
Provide the file rnase.mtz in the "Reflections" field. You can either browse for the file, or drag and drop it into the field.
A window will appear, asking you to select the columns to be imported from the MTZ file. In this case, the MTZ file contains only structure factor amplitudes, not intensities. Select F/SIGF and click "Apply".
A window will appear, allowing you to provide a description for the file. Click "OK".
Now re-supply rnase.mtz in the "Free R set" field. If you do not do this then a Free R set will not be used during refinement.
A window will appear, asking you to select the Free R column to be imported from the MTZ file. Select FREER and click "Apply".
A window will appear, allowing you to provide a description for the file. Click "OK".

That's all that's required in order to execute a round of refinement with REFMAC5, using default parameters.

Click the "Run" button on the top-right of the interface to run the job.

After a few seconds, the Results page will open, and will update after every refinement cycle. Notice that the current weight being applied to the X-ray term is displayed below the graph.

After the job has finished, the page will display a variety of information pertaining to the refinement.

Adjusting The Geometry Weight

By default, REFMAC5 automatically adjusts the relative weight between geometry and X-ray terms, updating the weight after each refinement cycle. For many cases it works sufficiently well. However, sometimes (especially at high resolution) the default weighting may give a relaxed geometry. In such cases, you can play with the geometry weight.

To adjust the geometry weight:

Click the "Clone job" button at the top of the interface. You will see that a second job has been created in the job list (on the left side of the interface).
Ensure the inputs are correct, and change the job title to something meaningful.
Select the "Restraints" tab.
Change the weight for "restraints versus experimental data" from "automatic" to "manual", and enter a weight (e.g. 2.0).
Rerun the job, and inspect the results once the job has finished.

What differences can you see between this refinement round and the previous refinement round that used automatic weighting?

Choosing an appropriate geometry weight

For low resolution data, you may want to use very small values - 0.01 or even smaller. At higher resolutions, this number may need to be as high as 10. You may need to run refinement multiple times using different values in order to get it right.

If after refinement the RMS bond distance is greater than 0.02 then you may want to reduce the weight, if the RMS bond distance is less than 0.01 then you may need to increase the weight.

A smaller weight means tighter geometry.

In order to know what weight to try, it is worth knowing the weight that REFMAC5 applies automatically. Then you can tighten or loosen the geometry weight by multiplying that value by e.g. 0.5 or 2, as appropriate. The weight that REFMAC5 applies can be found on the line directly below the graph on the Results page.

Inspecting the Electron Density

It is always important to manually inspect the electron density following refinement:

Click the "COOT" button at the bottom of the results page. This will create a new job (Job 3), which will allow the refined model and density to be displayed using Coot.
You do not need to change any options for Job 3. Click the "Run" button to launch Coot.
Can you identify any regions that require manual consideration?
Use Coot's "Unmodelled blobs" feature in the Validate menu to find the missing ligand.

When you are finished, open the "File" menu and select "Save to CCP4i2". This will pass your editted model back to CCP4i2 for use in further jobs. Open the "File" menu and select "Exit" to close Coot and return to CCP4i2.