ProSMART Tutorial - Part 3

Hydrogen Bond Type Secondary Structure Restraints
(For When There's No Good Homologous Model)

In this tutorial, we will demonstrate how ProSMART external restraints can be generated even in cases where a reliable high resolution homologue is not available for use as prior information. Specifically, we will look at hydrogen bond type restraints, which can be used to stabilise secondary structure (helices, sheets and loops). However, we will not cover structural fragment-based restraints, which can be used to restrain regions to particular conformations (e.g. an ideal alpha helix).

Since we have already covered the application of external restraints in crystallographic refinement in part 1, and analysis of refinement results in part 2, in this part of the tutorial we will focus more on restraint generation rather than application and analysis.

For consistency with previous parts of this tutorial, we will continue using the 3.5Å structure with PDB code 1ryx. A basic working knowledge of the CCP4i 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).

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

Generation Of Hydrogen Bond Type Restraints
Visualisation Of Hydrogen Bond Type Restraints
Generalisation To Generic Bond Type Restraints

Generation Of Hydrogen Bond Type Secondary Structure Restraints

ProSMART allows the generation of external restraints representing hydrogen bonding patterns. These restrain the distance between spatially-related nitrogen and oxygen atoms in the backbone. Such restraints may be generated for:

Helices (either all helices, or separately for 3₁₀, alpha, and pi helices);
Beta sheets;
Whole backbone (helices, sheets and loops).

ProSMART will detect candidate hydrogen bonds given the current conformation of the model, based on the proximity of main chain donors and acceptors. Consequently, the model must be in a relatively reasonable conformation in order for these restraints to work. These restraints are intended to help the model to adopt a more reasonable local backbone conformation, thus are useful when poor density would otherwise force the structure out of a sensible conformation.

Follow these steps to automatically generate external restraints and use them in refinement using the REFMAC5 CCP4i GUI:

Open the CCP4i GUI.
Open the REFMAC5 task interface, which can be found in the Refinement module.
Provide the 1ryx structure factors and coordinates in the "MTZ in" and "PDB in" fields.
In the "Use Prosmart" field, specify "to generate h-bond restraints".
Now open the "External Restraints" tab.
You will see that an option "Apply all Prosmart main chain h-bond restraints" has appeared, and is selected. If you leave this option selected, then restraints will be generated for all helices, strands and loops. Deselecting this option would allow you to limit the selection to specific secondary structure types.
However, for the purposes of this tutorial we will leave "Apply all Prosmart main chain h-bond restraints" selected.
Now run the job.

Remember to think about your refinement strategy

As discussed in part 1, proper thought should be given to selecting parameters in the "Refinement Parameters" and "External Restraints" folders in the GUI (e.g. number of cycles, use of hydrogens, use of jelly-body restraints, external restraints weight, Geman-McClure parameter, etc.). To avoid too much duplication, these considerations will not be discussed further in this tutorial.

Visualisation Of Hydrogen Bond Type Restraints Using PyMOL

It is always worth viewing software output in order to really understand what is going on, and check that behaviour is as expected/intended. Consequently, we will now use PyMOL to visualise the generated restraints representing hydrogen bonding patterns.

Follow these steps:

Looking at the CCP4i job list, double-click on the REFMAC5 job in order to open the qtRView results file (it doesn't matter whether the REFMAC5 job is "RUNNING" or "FINISHED"), and scroll down to the bottom of the page.
Click on the "Show" button next to "Prosmart analysis" in the "External Restraints" tab. This will 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". We will need to know the location of this directory in order to load the results into PyMOL for visualisation - 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 -> Run" and browse to the ProSMART output directory that you located earlier (in the ProSMART_Results.html page). From within this directory, select "Output Files -> Loader Scripts". You should see two files. Open the file called "1ryx_A_main_no_restraints.pml"

Learn to interpret the visualisation:

This script displays all of the generated hydrogen bond type restraints as dotted black lines between the atoms (or yellow lines depending on your version of PyMOL), and colours atoms according to the number of restraints that have been generated involving that atom:

White - no restraints;
Red - one restraint;
Blue - two restraints.

This representation is useful for intuitively identifying whether any hydrogen bond restraints are inappropriate (and thus should be removed), and whether there are any atoms that should be hydrogen-bonded but fall out of the detection range - this would tell you that you should fix the structure manually in Coot in order to satisfy hydrogen bonds before continuing with refinement.

Now let's look at a different representation:

Start by deleting all objects currently loaded (click the "A" button next to "all" in the menu on the right, and select "delete everything").
Select "File -> Run" and this time open "1ryx_A_main_residue_separation.pml".

Learn to interpret the visualisation:

This script displays all of the generated hydrogen bond type restraints as dotted red lines between the atoms (or yellow lines depending on your version of PyMOL), and colours residues according to the minimum residue separation between atoms involved in restraints:

White - no restraints;
Red - less than 3 residues (warning);
Light Blue - 3 residues (e.g. 3₁₀ helices).
Deep Blue - 4 residues (e.g. alpha helices).
Orange - 5 residues (e.g. pi helices).
Yellow - more than 5 residues (e.g. beta sheets).

This representation is useful for intuitively distinguishing implied secondary structure types, and determining whether hydrogen bond restraints correspond to the expected/intended residue separation.

Generalisation To Generic Bond Type Restraints

Further to generating bond type restraints representing hydrogen bonds, ProSMART allows a large degree of customisation, allowing users to automatically/systematically create generic bond type restraints according to some predefined criteria.

Customisation of generic bond type restraints is available from within the ProSMART CCP4i GUI:

Open the CCP4i GUI.
Open the ProSMART task interface, which can be found in the Structure Analysis module.
Select to "Align and generate restraints for target structure(s) using" -> "h-bonding patterns (e.g. secondary structure restraints)".
Provide the target model (1ryx.pdb) in the "Input target structure" field.
Open the "Generic or h-bond restraint parameters" folder.
To access the customisable generic bond type restraints functionality, select to "Apply generic/h-bond restraints to" -> "custom selection".
Finally, select "Customise restraint parameters" to allow customisation of target value and detection range.

Of course, all functionality is also available when using ProsAMART via the command line (see here for a list of ProSMART keywords).

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