ProSMART Tutorial - Part 2

Analysis Of Structural Changes That Occur During Refinement
(And Differences Between Homologues)

Here, we will build on part 1 of the tutorial, where we re-refined the 3.5Å model 1ryx using external restraints from the 2.15Å homologue 2d3i (completion of part 1 of the tutorial would be beneficial, but is not an essential prerequisite).

In this tutorial, we will use ProSMART to compare the the target (1ryx) and reference (2d3i) structures, analysing their structural conservation. We will then compare 1ryx and 2d3i against the model of 1ryx re-refined with external restraints, in order to assess the effects of the external restraints.

A basic working knowledge of the CCP4i GUI is assumed.

Before We Start

Required files:

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

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

1ryx_prosmart_refine.pdb - the model of 1ryx re-refined using external restraints from 2d3i.

Please click these three links to access the files and save them to a suitable location (e.g. your CCP4 working directory), or alternatively click here to get a .tar.gz containing all three files.

Execution Of ProSMART For Comparative Structural Analysis Using CCP4i
Navigation Of Output Files Using The ProSMART_Results.html Page
Choice Of Molecular Graphics Software

Execution Of ProSMART For Comparative Structural Analysis Using CCP4i

We will begin by using the ProSMART CCP4i GUI to compare the target (1ryx) and reference (2d3i) structures:

Open the CCP4i GUI.

Open the ``Run ProSMART'' task interface, which can be found in the Structure Analysis module.

Since we are not interested in generating restraints, select "Use ProSMART to align structures only".

Now provide the target (1ryx.pdb) reference (2d3i.pdb) models in the "Input target structure" and "Input reference structure" fields, respectively.

That's it!!! That's all that's required in order to run ProSMART for structural comparison, using default parameters. There are many options, but we will ignore them for now.

Run the job, close the ProSMART GUI window, and return to the main CCP4i job list.

Learn how to use command line ProSMART:

Note that we could have done this in a shell from the working directory using the simple command:

prosmart -a -p1 1ryx.pdb -p2 2d3i.pdb

Here, -a specifies that only alignment will be performed (i.e. restraints won't be generated; if -a isn't specified then both alignment and restraint generation will be performed), -p1 allows specification of the location of the target model(s), and -p2 allows specification of the location of the target model(s). To learn more, see the documentation; for a full list of keywords, see here.

The results will be located in a directory "ProSMART_Output" that will be created within the current working directory. This directory will contain the "ProSMART_Results.html" page that you can use to aid navigation of the output from within a web browser.

Navigation Of Output Files Using The ProSMART_Results.html Page

Looking at the CCP4i job list:

Double-click on the freshly-finished ProSMART job. This should open a simple qtRView results page.

You can view the ProSMART log file by selecting the "Log File" tab.

Back in the "Results" tab, click the "Show" button next to "ProSMART analysis". This will open the "ProSMART_Results.html" page in your default browser.

The ProSMART_Results.html page is designed to provide some information regarding the execution, and provide an easy way to navigate the results files output by ProSMART (this is better than trying to manually navigate the ProSMART_Output subdirectories, which can be a bit overwhelming).

The list of types of output that ProSMART can provide is shown on the left. Output types that are available for the current run are shown in blue text; those that are not available due to requiring different ProSMART operational modes/options are greyed out.

For this tutorial, we will limit our interest to ProSMART's superposition and residue-based pairwise colouring functionalities.

Learn how to use the ProSMART_Results.html page:

Quite simply, the ProSMART_Results.html page tells you what output is available, and tells you how to find the file you're looking for.

Try it out - click on "PDB Files" in the "Superposition" tab.

The "Chain Pair" list allows you to select one of the chain-pairs that ProSMART processed. In this case, there is only one compared chain-pair.
The "File" list allows you to select one of the files corresponding to the superposition of this chain-pair. In this case, there are five files:
- "1ryx_A" contains chain A from 1ryx, in its original coordinate frame.
- "2d3i_A" contains chain A from 2d3i, globally superposed onto 1ryx_A.
- "2d3i_A_Cluster0" contains chain A from 2d3i, superposed onto 1ryx_A using substructure 0.
- "2d3i_A_Cluster1" contains chain A from 2d3i, superposed onto 1ryx_A using substructure 1.
- "2d3i_A_Cluster2" contains chain A from 2d3i, superposed onto 1ryx_A using substructure 2.
The first two are always present; the latter are present only when rigidly conserved substructures between the compared chains are identified.
Click on one of the files in the "File" list.
This will either allow you to export the file or show the file in a window on the right, depending on your browser.
Importantly, the file's absolute path will be displayed in the upper-right portion of the window. If in doubt, this will always allow you to easily locate the file that you're looking for!

Choice Of Molecular Graphics Software

You may perform the remainder of this tutorial using CCP4mg and/or PyMOL for molecular visualisation:

Back to the tutorial main page