Results from ProSMART can be visualised using either CCP4mg or PyMOL.
In this tutorial, we will use PyMOL.
ProSMART generates PDB files and PyMOL colour scripts that are used to display the results of a ProSMART comparative structural analysis.
Various types of information are available for visualisation. Here, we will consider viewing substructure-based superpositions, colouring by local backbone conservation, and colouring by local side chain conformational dissimilarity.
Loading Structures Into PyMOL
First, load the PDB files output by ProSMART into PyMOL.
ProSMART needs to have been executed before we can load the results into PyMOL (see Part 2).
You should also know where the ProSMART results are located.
Assuming ProSMART has been executed using the ProSMART CCP4i GUI, follow these steps:
Open the ProSMART_Results.html page.
Now click on the "Input Parameters" button in the "Runtime Info" tab.
Look at the "Output Directory" field within "Program Directories". This directory contains all of the results from the ProSMART execution.
Now we are ready to load the results into PyMOL:
Open PyMOL.
Select "File -> Open".
Navigate to the ProSMART output directory, and select "Output Files -> Superposition -> PDB_files -> 1ryx_A_2d3i_A".
There will be five files in this directory:
1ryx_A.pdb - chain A from 1ryx, in its original coordinate frame;
2d3i_A.pdb - chain A from 2d3i, in the global coordinate frame of 1ryx;
1ryx_A_Cluster0.pdb - chain A from 2d3i, with the first substructure superposed;
1ryx_A_Cluster1.pdb - chain A from 2d3i, with the second substructure superposed;
1ryx_A_Cluster2.pdb - chain A from 2d3i, with the third substructure superposed.
Open all of these five files into PyMOL.
Now display all chains using the "cartoon" representation (in the "all" object in the bar on the right, select "S -> as -> cartoon").
To begin with, hide the three "Cluster" representations by clicking on the respective objects.
Viewing Substructure Superpositions - Analysis Of Global Conformational Changes
We will now compare the target (1ryx) and reference (2d3i) structures, looking at domain motion in order to gain intuition regarding differences in their global conformations:
Hide "2d3i_A", and show "2d3i_A_Cluster0".
The structures now seem better superposed, but it's not that easy to see which bits are superposed and which aren't. Colouring can help with this.
Go to "File -> Run", and navigate in the ProSMART output directory to "Output_Files -> Colour Scripts -> 1ryx_A_2d3i_A".
This directory contains a variety of colour scripts that you can use to colour your structure for various purposes.
Run the script "cluster_0.pml", and wait for PyMOL to process the script.
The residues are now coloured according to the similarity of their local coordinate frames.
Residues that closely relate to the rigid substructure/cluster are coloured red, gradually fading to white for regions that adopt a noticeably different global conformation.
We can see that the core of the structure seems relatively well-conserved between the two homologous chains, although it is hard to tell how well-conserved the other two domains are...
We will look at the other two domains/substructures/clusters:
Hide "2d3i_A_Cluster0", and show "2d3i_A_Cluster1".
Now go back to "File -> Run". This time run the script "cluster_1.pml".
Here, the rigid substructure is coloured green. We can now see that this domain seems relatively well-conserved, despite the large domain motion.
Now let's look at the third substructure:
Hide "2d3i_A_Cluster1", and show "2d3i_A_Cluster2".
Run the script "cluster_2.pml".
This time, the rigid substructure is coloured blue. Zoom in and rotate the structure, looking at the rigid substructure. Can you see any regions (e.g. loops) that seem to belong to the domain but show conformational differences between the two compared structures?
Again this domain seems relatively well-conserved. However, "relatively well-conserved" isn't very scientific... we need to look at a more detailed analysis of structural conservation at the local level.
Local Structural Conservation Of The Backbone
Hide "2d3i_A_Cluster2" and show "2d3i_A_Cluster0".
We will colour the structures according to the "Minimum" score, which is a measure of local backbone conservation - it is not dependent on how you superpose the structures.
Go to "File -> Run", and run the script "minimum.pml".
Residues in a similar local environment are coloured yellow, gradually changing to red indicating comparative structural dissimilarity.
In order better understand backbone conservation, it is often useful to alter the colour gradient, depending on the overall local structural conservation of the compared chains.
Whilst this can be achieved dynamically in CCP4mg, ProSMART must be re-executed in order to change the colours in PyMOL (i.e. the colour script must be recreated):
Go back to the CCP4i GUI. Highlight the ProSMART job that we ran earlier, and click the "ReRun Job" button.
Now open the "Output options" folder. This contains various options for customising the way in which ProSMART generates results for viewing in PyMOL (e.g. you can change the colours from yellow/red to something else if you want).
Click on the selection box for altering the colour gradient.
We will now change the gradient for mainchain scores to "1.5". This value is the score threshold representing complete dissimilarity (red).
Now run the job.
When ProSMART has finished, we can return to PyMOL:
Once again, go to "File -> Run", and run the script "minimum.pml".
Local Structural Conservation Of Side Chains
For regions of the structure that sufficiently conserved in terms of backbone conformation, we are often interested in whether side chains maintain their conformation relative to the backbone despite any conformational changes such as domain motion.
We will now consider the "Side Chain RMSD" score, which colours residues according to the RMSD of the corresponding side chain atoms after superposition of the local backbone:
Run the script "sidechainRMSD.pml".
Since we're comparing side chains, we should show the side chains. Next to the "all" object, select "S -> lines".
(you may also want to display the caroon representation, for clarity)
Now we can see which side chains adopt a similar conformation in the compared chains (yellow), and which adopt a dramatically different conformation (red).
We can see that many of the side chains adopt different conformations in the two models. Some of these differences may be in the crystal, whilst others may be due to incorrect modelling.
Analysis Of Structural Changes That Occur During Crystallographic Refinement
So far in this tutorial we have focussed on comparing the target (1ryx) and reference (2d3i) structures.
We will now consider the comparison of the target structure before (1ryx.pdb) and after (1ryx_prosmart_refine.pdb) refinement with external restraints (data files were made available at the start of part 2).
Firstly, follow these steps:
Start a new session - close PyMOL, then re-open it.
Start a new ProSMART CCP4i job, and load 1ryx.pdb and 1ryx_prosmart_refine.pdb as the target and reference structures, respectively.
Change the main chain score colour gradient to 1.5.
Run the job.
Open the two ProSMART PDB files 1ryx_A.pdb and 1ryx_prosmart_refine_A.pdb in PyMOL.
Change the view mode to "cartoon".
The first thing to notice is that the global conformation of the model didn't change during refinement, despite the use of external restraints to a homologous structure that is in a different conformation!
Now we'll investigate whether there have been many changes to the backbone:
Colour the structures using the "Minimum" score (using the script "minimum.pml").
Zoom in and have a good look at the structure; see which regions have seen large changes to the backbone during refinement and which haven't.
Now investigate how much side chain conformations changed:
Colour the structures using the "Side chain RMSD" score (using the script "sidechainRMSD.pml").
Add side chain "lines" to the representation.
Zoom in and have a good look at the structure; see which side chains have changed during refinement and which haven't.
Here, a large number of the side chains have changed conformation, indicating that a lot of manual inspection and model rebuilding in Coot would be required in this case.
In other cases, fewer side chains may have changed conformation - this information can be helpful in speeding up manual model building in Coot.
Assessing Influence Of Reference Structures During Externally-Restrained Refinement
So far in this tutorial we have compared the target and reference structures, and compared the original and re-refined models.
We will now consider the comparison of the re-refined model (1ryx_prosmart_refine) and the reference structure (2d3i).
This will illustrate the influence of the external restraints.
Similarly to above, follow these steps:
Start a new session - close PyMOL, then re-open it.
Start a new ProSMART CCP4i job, and load 1ryx_prosmart_refine.pdb and 2d3i.pdb as the target and reference structures, respectively.
Change the main chain score colour gradient to 1.5.
Run the job.
In PyMOL, open the ProSMART PDB files: 1ryx_prosmart_refine_A.pdb, 2d3i_A_Cluster_0.pdb, 2d3i_A_Cluster_1.pdb and 2d3i_A_Cluster_2.pdb.
Hide 2d3i_A_Cluster_1.pdb and 2d3i_A_Cluster_2.pdb, and change the view mode to "cartoon".
Colour the structures by the "Minimum" score to assess backbone conservation.
The first thing to note is that the target structure has been necessarily pulled towards the reference structure during refinement - the two structures have locally-similar backbones.
However, there are a few regions that have not been pulled into the conformation of the reference structure.
The analysis suggests that overall the backbone of the re-refined model is more locally similar to the reference structure than to the original target model.
Now investigate whether there have been many changes to the side chains:
Colour the structures using the "Side chain RMSD" score (using the script "sidechainRMSD.pml").
Add side chain "lines" to the representation.
Zoom in and have a good look at the structure; see which side chains have changed during refinement and which haven't.
There are a substantial number of side chains that adopt different conformations in the re-refined and reference models.
This demonstrates that the external restraints have not pulled these side chains out of their conformations - good density should help to keep structure in place.
If not, it could be that you need to loosen the external restraints weight.
Summary
External restraints can pull regions into incorrect conformations, particularly when density is weak.
Whilst this sounds like a bad thing, it is not! Indeed, this often has the positive effect of revealing new features in the density, and causing difference density to appear in regions where the "true" model should be placed (where the word "true" is used tentatively...).
If this happens, and after manual inspection in Coot you conclude that the external restraints have had a negative effect in a particular region, then you should exclude the region in question when generating the external restraints.
Alternatively, you can apply the corrections manually in Coot, before continuing subsequent rounds of refinement with jelly-body restraints.