Practical: Molecular Graphics

Aims

• To reinforce the basic concepts of protein geometry described in the lectures.
• To give practice in using molecular visualisation software.

Objectives

After this practical you will:

• be competent in performing simple interactive tasks with the RasMol molecular visualisation program;
• be able to identify protein main chain atoms and recognise some amino acid residue side chains.

Introduction

There are many programs, both freely available and commercial, for molecular visualisation and modelling. Since these have been written for different primary purposes, they have different strengths and weaknesses. RasMol is good for molecular visualisation and rendering molecules in different styles. If you need help during the practical, please ask. If you need help when working on your own, a useful starting point is the RasMol Manual.

We shall view protein structures taken from the Protein Data Bank (PDB), which is the worldwide repository for 3-D biological macromolecular structure data. A good structure to start with is crambin (PDB Id: 1CRN). Crambin is a small protein and therefore convenient to explore. It contains examples of alpha-helices, a beta-sheet and disulphide bonds. We shall then look at some larger proteins, and examine interesting aspects of their structures.

Exercises

1. Download the structure of crambin from the Protein Data Bank web site:

   1. Go to the Protein Data Bank web site and "SEARCH" for the structure with PDB ID "1CRN".
   2. Click on "Download Files" menu near the top right corner of the browser window.
   3. Click on "PDB File (Text)" and save the file to disc.

   Run the RasMol program by typing rasmol in a terminal window.
   If this does not work, please read these notes.

   Zoom in on part of the protein chain (Shift-Left) and work your way along the protein chain identifying some amino acid residues.

2. Hydrogen bonds can be displayed in Rasmol. Select only the main chain atoms by typing "restrict mainchain", then display main chain hydrogen bonds by typing "hbonds on". Look at the main chain hydrogen bonding pattern in the helical regions. The C=O group of the residue at position i forms a hydrogen bond with the N-H group of which other residue?
   Look also at the hydrogen bonds in the region of beta-sheet.

3. Measure the distances between the sulphur atoms in the disulphide bridges. To start measuring distances in RasMol you need to type "set picking distance" before picking atoms.

4. Measure the omega dihedral angle between a few pairs of residues. To start measuring dihedral angles in RasMol you need to type "set picking torsion" before picking atoms.

5. Protein Data Bank entry 1TLD is a high resolution structure of bovine beta-trypsin, which is a serine protease (a "protein-cutting machine"). His57, Asp102 and Ser195 are important active site residues in serine protease enzymes. Locate these three residues and draw a sketch showing the distances from the nitrogen atoms in the side chain of His57 to the oxygen atoms in the side chains of the other active site residues.

   To see the side chains of these residues more easily, display the whole structure as a wireframe model, then select the three side chains:

         RasMol> select (57, 102, 195) and sidechain
       

   Select "Ball & Stick" from the Display menu to highlight these side chains.

6. Protein Data Bank entry 3BTK is a complex between bovine beta-trypsin and bovine pancreatic trypsin inhibitor (BPTI). Is the active site of beta-trypsin accessible to a substrate? Try the following sequence of RasMol commands:

         RasMol> colour chain
         RasMol> select 57e,102e,195e
         RasMol> colour cpk
         RasMol> spacefill
         RasMol> select not hoh
         RasMol> spacefill
       

7. Serpins are a family of serine protease inhibitors whose mechanism is different to that of BPTI. Protein Data Bank entry 1ATU is the active form of human alpha-1-antitrypsin, a serpin. Look at this structure, and locate residues Met358 and Ser359. These residues are in the reactive-centre loop, and the reaction with a serine protease cleaves the loop between these two residues. Describe the structure of the main chain between Lys343 and Met358.

8. Protein Data Bank entry 1EZX is a serpin-protease complex. The molecules present are human alpha-1-antitrypsin (a serine protease inhibitor, or serpin) and bovine trypsin (a serine protease). The peptide bond between Met358 and Ser359 of alpha-1 antitrypsin has been cleaved. Measure the distance between the main chain carbon atom of Met358 and the nitrogen atom of Ser359. Describe the structure of the main chain between Lys343 and Met358. Locate the active site residues of the trypsin molecule.

9. The DSSP program can be run by typing the command dssp in a terminal window. The name of a Protein Data Bank file should be given as a command line argument, e.g. dssp 1CRN.pdb

   Look at the output from DSSP for Protein Data Bank entry 1CRN. Look in particular at the column with secondary structure assignment codes, and compare with the structures that you see in RasMol.

Supplementary Material

In October 2003 Trypsin was featured as the Protein Data Bank's Molecule of the Month and was the European Bioinformatics Institute's Protein of the Month. In May 2004 Serpins were featured in the Molecule of the Month series.

You can find out more about the serpin-protease complex in the article describing the structure:

• Huntington, J.A., Read, R.J., Carrell, R.W. (2000) Structure of a Serpin-Protease Complex Shows Inhibition by Deformation, Nature, 407, 923-926 (PubMed)

The mechanism by which serine proteases cleave peptide bonds is illustrated in this animation.

An interesting historical perspective on developments in molecular visulisation has been written by Martz and Francoeur.