REFMAC5/ProSMART Tutorial

Restraining a Low-Resolution Structure
Using A Homologous Higher-Resolution Model

In this tutorial, we will generate ProSMART external restraints for the deposited 3.5Å structure with PDB code 1ryx using the sequence-identical 2.15Å structure 2d3i, which adopts a different global conformation and was solved in a different space group.

A basic working knowledge of the CCP4i2 GUI is assumed.

Before We Start

Required files:

1ryx.pdb - the deposited low-resolution model;

1ryx.mtz - the deposited structure factors (coverted to MTZ format);

2d3i.pdb - the deposited high-resolution model that will be used as a reference structure.

Please click these three links to access the files and save them to a suitable location on your computer.

Attempt Standard Refinement
Jelly-Body Refinement
Refinement using ProSMART External Restraints
Compare Results
Manual Inspection in Coot

Attempt Standard Refinement

We will begin by attempting to refine the structure without using additional regularisers:

Click on the "Task menu" at the top-left of the CCP4i2 interface, and navigate to "Refinement - REFMAC5".
Give the job a meaningful title, e.g. "Tutorial Example 4 - Low Resolution", and ensure that data from a previous job are not used to populate the input fields.
Provide the files 1ryx.pdb and 1ryx.mtz as inputs in the "Atomic model", "Reflections", and "Free R set" fields.
Set the "Number of refinement cycles" to 20, and untick "Use hydrogens during refinement".
Click "Run" to start the job, and continue with the tutorial while this job runs....

Jelly-Body Refinement

Since only low-resolution data are available, additional regularisers are required in order to stabilise refinement. Now try refining the structure using jelly-body restraints:

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 4 - Jelly Body".
Click on the "Restraints" tab, and select "Use jelly-body restraints".
Click "Run" to start the job, and continue with the tutorial while this job runs....

Refinement using ProSMART External Restraints

Whilst jelly-body restraints can stabilise refinement, they can't help the model to escape local minima and move into a more sensible conformation. However, this can be achieved using external restraints from a homologous structure. We will now refine the structure using ProSMART external restraints from a homologous model (2d3i):

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 4 - External".
Click on the "Restraints" tab and deselect "Use jelly-body restraints", as we do not want to use jelly-body restraints and external restraints at the same time.
Provide the file 2d3i.pdb as input in the "Reference model" field. This will cause ProSMART to be executed to generate external restraints for 1ryx using structural information from 2d3i, prior to executing REFMAC5 to refine the model.
Click "Run" to start the job....

Compare Results

Compare the results from the three different refinement runs.

Manual Inspection in Coot

Now inspect the model and density after refinement using external restraints:

Click the "COOT" button at the bottom of the results page. This will create a new job, which will allow the refined model and density to be displayed using Coot.
You do not need to change any options for this job. Click the "Run" button to launch Coot.
Can you identify any regions that require manual consideration?
Increase the map radius to 20Å (Edit -> Map Parameters -> Map Radius), and show symmetry related molecules (Draw -> Cell and Symmetry -> Show Symmetry Atoms).
Go to residue 475.
It is clear that this region is is need of rebuilding. Attempt to improve the model in this region using real-space refinement.
Now we want to save the editted model, and attempt re-refinement using REFMAC5.
Save the model to CCP4i2 from Coot (File -> Save mol to CCP4i2), and then exit Coot (File -> Exit).
This model is now available for further re-refinement by REFMAC5 from within CCP4i2.
Now try re-refining this model using jelly-body restraints, inspect the refinement statistics, and inspect the model and density in Coot.

(Optional) Generating And Using External Restraints Via The Command Line:

(Warning - only recommended for those who like using the command line!)

Whilst it's very convenient to use the CCP4i2 GUI for running jobs, it's a good idea to also gain experience in running programs via the command line in order to really understand what's going on.

Open the command line prompt, and navigate to the directory containing the data files 1ryx.pdb, 1ryx.mtz and 2d3i.pdb.
To generate the external restraints, type:
```
prosmart -p1 1ryx.pdb -p2 2d3i.pdb
```
and hit ENTER. This command specifies that ProSMART should generate restraints for 1ryx (keyword: -p1) using the homologous structure 2d3i (keyword: -p2). A list of other keywords can be found in the ProSMART documentation.
If successful, a directory called "ProSMART_Output" has been created, and the final external restraints file, called "1ryx.txt", has been created within this directory.
Create a simple script specifying the command to run REFMAC5, with the appropriate keywords:
```
refmac5 > refmac_log.txt \
    XYZIN 1ryx.pdb  \
    HKLIN 1ryx.mtz  \
    XYZOUT pdb_out.pdb \
    HKLOUT mtz_out.mtz \
<<EOF
MAKE -
    HYDROGEN NO -
    NEWLIGAND CONTINUE
NCYC 40
@ProSMART_Output/1ryx.txt
MONI DIST 1000000
END
EOF
```
The "EXTERNAL" keywords specify options that will be applied to any external restraints specified below that point in the script. The "@ProSMART_Output/1ryx.txt" line tells REFMAC5 to use all external restraints that are in our restraints file. The "MONI DIST 1000000" keyword avoids any restraint outliers being recorded as warnings in the REFMAC5 log file.
Run this script in order to execute REFMAC5.

It is recommended to use external restraints in early/medium stages of refinement, followed by subsequent rounds of jelly-body refinement (without external restraints). Ultimately, the crystallographer should decide what strategy makes the most sense in their particular case. Further to injecting external information obtained from reliable sources (e.g. homologous structures), external restraints can be used to help stabilize a desired conformation whenever the density alone is of too poor quality to maintain it.

Back to the tutorial main page

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Disclaimer - it is important to note that we are not criticizing the authors of the original deposited low-resolution model. Rather, due to improvements in macromolecular crystallographic refinement tools in recent years, we are now able to gain further insight that was not routinely possible at the time of original structure solution. Furthermore, we do not imply that the modifications applied to the model during this tutorial are necessarily accurate – this could only be confirmed/re-evaluated after performing extensive model rebuilding and refinement.