Aims

• To reinforce the importance of macromolecular structure in understanding biological function.
• To give practice in using molecular graphics software.
• To give practice in interpreting a contact map.

Objectives

After this practical you will:

• have used the RasMol molecular visualisation program;
• be competent in using RasMol to locate functionally important amino-acid residues in experimentally determined macromolecular structures;
• be able to recognise domains and interacting elements of secondary structure in contact maps.

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.

Exercises

1. Protein Data Bank entry 2CSN contains the structure of Casein Kinase-1 from Schizosaccharomyces pombe.

   1. Study the contact map for this protein kinase. Identify structural domains and contacts between elements of secondary structure.

   2. Look at the structure of 2CSN and correlate features identified from the contact map with the three-dimensional structure.

2. Protein Data Bank entry 1GD2 is a bZIP transcription factor bound to DNA.

   1. Highlight the residues of the transcription factor that make contact with the DNA bases.

   2. What is the sequence of the DNA molecule in this structure? Is it significant that the DNA fragment is a self-complementary palindrome?

   3. The transcription factor in this structure is an example of a "leucine zipper" protein - one of the largest families of transcription factors in eukaryotic cells. Why is the name "leucine zipper" appropriate when describing this protein?

3. Protein Data Bank entry 1G7C is a yeast eEF1A-eEF1Balpha complex, with a non-cleavable GTP analogue bound to eEF1A.

   1. Locate residue Lys205 in eEF1Balpha.

   2. Discuss the consequences of mutating Lys205 in eEF1Balpha to alanine.

4. Protein Data Bank entry 1BL8 is a potassium channel protein from Streptomyces lividans.

   1. Locate the potassium selectivity filter. The residues involved are listed in Swiss-Prot entry KCSA_STRCO. Which protein atoms make contact with potassium ions?

   2. The four Tyr residues from the selectivity filter interact with eight Trp side chains from the pore helices to form a massive sheet of twelve aromatic residues. Locate these residues in the structure. Note the hydrogen bonding between the Tyr hydroxyls and Trp nitrogens.

5. (For those who want some programming practice.)
   Write a prorgam that can read a Protein Data Bank file and generate a contact map.

   A simple approach is to make use of the "dotplot" program used in Exercise 4 on the "Perl 2" exercise sheet from the Programming Tools course. You can write a program that reads the coordinates of "CA" atoms in a PDB file then, for each pair of CA atoms, writes out a pair of residue numbers if the distance between the CA atoms is less than a threshold. The Tcl/Tk program /users/mdstud/kemp/ptools/perl/perl2/dotplot.tcl can then be used to plot the points as a contact map.

6. (For those who want more programming practice.)
   Write a program that can read a Protein Data Bank (assume that it contains one chain, like Protein Data Bank entry 1CDH) and identify the residue at which the chain is most clearly split into two parts/domains. Your program should use the simple scoring function that is used in the DOMAK program: (int_A/ext_AB)*(int_B/ext_AB)