Aims

• To reinforce the basic concepts of protein structure 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;
• understand how main chain conformation is represented on a Ramachandran plot.

Introduction

In this practical you will use the RasMol molecular visualisation program. 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), which contains examples of alpha-helices, a beta-sheet and disulphide bonds. We shall then look at myoglobin.

Exercises

(A) Crambin

1. Download the structure of crambin from the Protein Data Bank web site. Zoom in on part of the protein chain and work your way along the protein chain identifying some amino acid residues.

2. 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.

3. 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.

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.

(B) Myoglobin

1. Protein Data Bank entry 1MBN is an X-ray structure of sperm whale myoglobin.

   Start with a wireframe representation of the whole molecule, then highlight the helix between residues 20 and 34 as follows:

         RasMol> select 20-34
         RasMol> cartoons
         RasMol> select 20-34 and sidechain
       

   and select "Ball & Stick" from the "Display" menu.

   Looking along the axis of this helix, describe the distribution of charged and uncharged residues.

   You might find it convenient to reset the center of rotation by typing "set picking center" then clicking on an atom, and to colour the residues according to their properties e.g. by typing "colour amino".

   Next, highlight the same helix as follows:

         RasMol> select all
         RasMol> colour white
         RasMol> spacefill
         RasMol> select 20-34
         RasMol> colour amino
       

   Which side chains of this helix are accessible to solvent?

2. Again, start with a wireframe representation of sperm whale myoblobin (1MBN). Highlight the region between residues 20 and 42 as follows:

         RasMol> select all
         RasMol> cartoons
         RasMol> select 20-42
         RasMol> colour red
       

   Describe the conformation of this region. In the previous question you looked at the region between residues 20 and 34; explain what happens to the conformation immediately after that region.

3. Measure the phi and psi angles of some residues in the middle of alpha helices. Where are these located on a Ramachandran plot?

4. This protein contains four proline residues. Where are these located on a Ramachandran plot?

5. Where are the residues at positions 80 and 150 located on a Ramachandran plot?

6. Read the description of Ramachandran plots by residue type in the PROCHECK Operating Manual. Discuss whether the residues examined in Questions 4 and 5 have favourable main chain conformations.

Supplementary Material

PROCHECK results and various other information about each Protein Data Bank entry are given in PDBsum.