In this tutorial, we will use REFMAC5 to refine the model of a molecular chaperone with PDB code 1n5b, treating each chain as a separate TLS group.
The asymmetric unit contains two dimers, chains A + B and chains C + D.
Data is in P212121, collected to 2.0Å.
A basic working knowledge of the CCP4i2 GUI is assumed.
Start by loading the model and data into CCP4i2, and running simple refinement:
Click on the "Task menu" at the top-left of the CCP4i2 interface, and navigate to "Refinement - REFMAC5".
A new job will be created.
The main inputs may already be populated with data from a previous job, in which case select "No" from the "Use data from job" selection box.
Give the job a meaningful title, e.g. "Tutorial Example 2 - TLS Refinement".
Provide the files 1n5b.pdb and 1n5b.mtz as inputs in the "Atomic model", "Reflections", and "Free R set" fields.
Click "Run" to start the job.
Continue with the tutorial while this job runs...
Automatic TLS Refinement
Now re-run the job, this time enabling automatic TLS refinement:
Click on the "Clone job" button at the top of the interface. This will create a new job, pre-populated with the inputs from the previous job.
Give the job a meaningful title, e.g. "Tutorial Example 2 - AutoTLS".
Click on the "Parameters" tab, and select "automatic" from the TLS parameters dropdown box. This will enable automatic creation of TLS groups (one for each chain). TLS parameters will be refined prior to the main rounds of restrained refinement.
Click "Run" to start the job, and continue with the tutorial while this job runs...
Automatic TLS Refinement with Custom Weight
In this case, the model geometry is not very stable during refinement, as can be seen in the graph of rmsBonds per cycle. This is due to the auto-weighting changing the geometry weight during the refinement process. Consequently, we shall now re-run the job using a fixed weight.
Click on the "Clone job" button at the top of the interface. This will create a new job, pre-populated with the inputs from the previous job.
Give the job a meaningful title, e.g. "Tutorial Example 2 - AutoTLS, weight 0.1".
Click on the "Restraints" tab, and change the X-ray/geometry weight to 0.1.
Click "Run" to start the job, and continue with the tutorial while this job runs...
TLS Refinement using pre-defined TLS groups from TLSMD
So far we have only used TLS groups as automatically specified by REFMAC5. It is possible to specify custom groups for TLS parameter refinement.
It is possible to manually specify TLS groups (see below), as well as TLS parameter estimates.
One automated solution is to use the TLSMD server (http://skuld.bmsc.washington.edu/~tlsmd/).
The file 1n5b_tlsmd.tls was created using the TLSMD server.
We shall now re-run the job using these custom TLS coefficients, still using the weight 0.1.
Click on the "Clone job" button at the top of the interface. This will create a new job, pre-populated with the inputs from the previous job.
Give the job a meaningful title, e.g. "Tutorial Example 2 - TLSMD, weight 0.1".
In the Parameters tab, select "explicit TLS group definitions" in the TLS parameters dropdown box, and provide the 1n5b_tlsmd.tls file in the "TLS coefficients" field.
Click "Run" to start the job.
Compare Results
Consider the results from the four different refinement runs.
How do they compare?
Why do the runs involving TLS refinement start with such high R-factors?
What are the pro's and con's of each refinement protocol?
Which refinement protocol is the best, in this case?
Extra Information
Note that it is possible to create/import TLS group definitions using the "Import and/or edit TLS set definitions" task in the CCP4i2 interface.
For the analysis of TLS parameters, see the program: TLSANL.