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Tutorial Two: Relax Structure, Scatter Plot, Minimize Structure

Estimated time: 30 minutes

This tutorial will instruct users how to generate optimized (low energy) protein conformations using the Relax and Minimize Actions, and the Scatter Plot tool.

In order to run this tutorial, you will need to know how to use the tools from Tutorial One.

(Caveat: The estimated time does not include the time it takes for jobs to run on the server. There will be several pauses while you wait 20 minutes or more for structures to run on the cloud before you can move on to more steps.) 

1) Introduction to Running Relax

Relax is an aggressive Rosetta tool for sampling multiple conformational changes in a protein to find a structure with the lowest Rosetta Energy. To read more about Relax click here. Relax is the best way to find the lowest energy conformation for your structure. Since all our tools will use energy for optimization, using the lowest energy structure for those tools will decrease the noise and improve their accuracy.

To find the structure with the lowest energy, multiple rounds of Relax will be run. The scores for the structures will decrease as more low energy structures are generated and the range of scores will narrow. After each round of Relax the best structure or structures will be selected to use as the starting structures for another round of Relax. When the scores of structures can no longer get lower, an optimized structure has been generated (which is the structure with the lowest energy).

To run Relax, first select a Project or Create a Project and load the structure 5KIZ. In Tutorial One we recommended running Prepare on a new structure after loading it into Bench. However Relax is a more aggressive version of Prepare so we can skip it.

After the structure has loaded, click on the 5KIZ folder in the left window in order to bring it into the center tab.

Click the  to the left of the structure name in order to select it. Then, in the right window, click  . This will open a Relax tab in the center window. Enter  and click .  (For this tutorial, we only have you run 5 repeats to limit your credit usage, but we normally recommend 20 repeats in order to get enough diversity in your results to make significant improvement).This should take 20 minutes or more depending on how many jobs are in the Queue ahead of it.

Once the Relax has run, it will appear in the left window. Click the  to the right of the Relax results file  and change the name to relax 1 5KIZ. Click on the Relax folder in order to open it’s tab in the center window. Structures will be listed in the order that they were created and labelled structure one to structure five. You can reorder the structures by any of the columns:

Click on . The structure with the lowest energy will  be listed first. In the example run shown below the structure with the lowest energy had a score of  -187.43:

Click  again to reverse the order of scores; the structure with the highest energy will now be listed first.

2) Selecting One or Multiple Low Energy Structures with Scatter Plot

We will run multiple rounds of Relax. After each Relax, we will select the best structure or structures to use as the starting structures for another round of Relax. You will check the energy for each round of Relax. After each round of Relax, you should expect the score to trend lower, but the range to become more narrow. You know that you have achieved the energetic minimum when the score stops going down. But first, we will look at the results of this Relax run in the next section.

Before we go on, why did we run multiple rounds of Relax?

Running Relax is a way to make aggressive changes to the conformation in order to find new conformations that are more favorable. Many changes are sampled during Relax in order to find ways to improve the structure. Relax will always sample the same number of conformations. Sometimes, this is not enough sampling to find the lowest score, so we need to keep sampling more conformations in order to find the best one. In this case, use the results of Relax as a starting structure to run more Relax repeats. If Relax cannot find a better conformation, it will give you a Relaxed conformation with essentially the same score as the starting structure.

2) Analyze Scatter Plo

While you are still in the Relax tab for your finished Relax, click  in the lower center window. Change the button to . By default, Scatter Plot displays the Score on the Y axis and the RMSD (compared to the starting structure) on the X axis:

However, you can change either of these axes to Score, RMSD, or to individual energy terms. From the default Scatter Plot setting, select a few structures (3 or 4 structures) that have the lowest energy, but have a broad range of RMSD. You can do this by click & dragging a box around the dots that are lowest in the plot:

These selections will turn blue. You can work with these selections by clicking either of the icons highlighted with red circles in the image above. Click  for the selected structures in the plot to become checkmarked in the list above. We will use these structures for the next round of Relax because they are energetically favorable and structurally diverse. This is best done from the Scatter Plot view so that you can be certain that you are selecting a diverse sampling. Before starting the next round of Relax, click on the  in order to visualize the areas of structural diversity. This activates the  above for these structures. Click  and select :

You can see (above) that the major differences in my structures are in two loop regions and another section which is mostly beta sheet.

3) Find structure’s Energetic Minimum with Relax, then Minimize

You should have three or four structures checkmarked from the previous step. We will use these as starting structures for Relax iterations in order to find the energetic minimum. Click  to open a Relax tab in the center window. You will note that my four structures that are starting structures are listed (below).

Enter  because each structure will repeat 50 times, totalling 200. Click . Once the Relax has run, it will appear in the left window. Change the results file name to relax2 5KIZ. Click on the folder in order to analyze the results.

As mentioned before, we expect the lowest energy to get lower and the energy range to be more narrow. Select the lowest energy structure and make another round of Relax with 50 repeats. Keep doing this with the lowest energy structure until the lowest energy structure is no longer an improvement. Once you have reached the minimum, select that structure and click , then . Minimize will always have only 1 final structure, so you do not have to make repeats. For more information on Minimize, click here. Change the results file name to Optimized 5KIZ.