University of the Witwatersrand, Johannesburg
		Wits Bioinformatics

Protein Structure Exploration with
Ligands Sites Explorer & Rasmol
Summer University 2011

Practical and Assignment

Dr.Abdelkrim Rachedi

In the practical session you will learn how to use the Ligands Sites Explorer site and Rasmol programme to explore
all the levels of protein structure; secondary, tertiary and quaternary. You'll also get insight on protein
function by visualizing ligands and their environment.

Assignment questions are denoted in red colour font.

Ligands Sites Explorer:

Ligands Sites Explorer (LSE) is an online tool that allows exploration and visulization^(*) of protein
structures information and associated ligands. LSE is the creation of the Wits Bioinformatics (see Services).

LSE is accessed at: http://emboss.bioinf.wits.ac.za/lse/

(*) The LSE visualization module uses the Jmol applet through a locally developed wrapper code.

RasMol:

The name RasMol comes from raster display of molecules which is a type of computer display useful for
showing solid surfaces.

Rasmol was created by Roger A. Sayle of Glaxo Corporation and the University of Edinburgh, Scotland.

Rasmol can be downloaded from: Rasmol Quick Start

A- LSE: Exploration of Seconday, Tertiary and Quaternary structure levels:

• Using LSE, explore the protein structures in the following table:

  File	Structure ID	Protein Name	Bound Ligand
  3dfr.ent	3dfr	Dihydrofolate Reductase	MTX
  1gzx.ent	1gzx	Haemoglobin (oxygenated state)	HEM, OXY
  . The main data type contained in these files is the atomic coordinates X, Y and Z of the protein structure, any ligands and water molecules.



• Click on the link 3dfr from the table above.
  This will launch LSE with information about the 3dfr structure entry.
• Read the information provided by the LSE tool.
• Explore the Ramachandran plot. What does the plot tell you about the structure?
• From the top left-hand LSE window, click the link 3dfr (3D-view the whole entry).
  This will display the 3D-structure of the protein.
• Once the structure is displayed, click the link "Mouse Controls & Measurments",
  found in the display window, for related information.
• Helices are shown in red.
  β-stands (members of the central &beta-sheet) are shown in yellow.
  Silver coloured are the loop regions.
• From the Scene Rendering: options, use Cartoons, Trace and Rockets for more exploration
• Use the option H Bonds to expose the net of H-bonds holding the Helices and Stands/Sheet to conformation.

What do you think the whole scene is showing?

• Click on the link 1gzx from the table above.
• Read the information provided by the LSE tool.
• Explore the Ramachandran plot. What does the plot tell you about the structure?
• From the top left-hand LSE window, click the link 1gzx (3D-view the whole entry).
  This will display the 3D-structure of the protein.
• From the Scene Rendering: options, use Cartoons, Trace and Rockets for more exploration.
• From the Scene Colour: list options, select chain.
  This means that we are asking Jmol to colour the structure component chains in different colour each.

How many chain does this proteins is made of?

What do you think the whole scene is showing?

B- Rasmol: Exploration of Seconday, Tertiary and Quaternary structure levels:

Notes:
• "File: Open" means click on Rasmol menu File then click Open
  Other menu usage will fellow the same scheem & denoted in bold font.
• When Rasmol first time open a PDB file, the mode wireframe is used by default.
• To Rotate the scene, Hold down mouse left button & move mouse.
• To Zoom in the scene, Hold down both Shift key & mouse left button then drag mouse down.
• To Zoom out the scene, Hold down both Shift key & mouse left button then drag mouse up.
• To Translate the scene, Hold down mouse right button then drag mouse left or right
  depending on where you want the molecule to be translated to.
• "<r>" stands for Return the keyboard entry
• Rasmol> "command" "<r>": means type a Rasmol cammand at the Rasmol Command line.

Download Structure entries:

• Download both structure files 3dfr.ent and 1gzx from the IBT_MCB_2011 site http://www.bioinf.wits.ac.za/IBT_HGMM_2011/Other_Material/.
• Save the files in a diretory in the desktop.

Launch RasMol:

• Windows users:Double click on the Rasmol icon from your windows desktop.
• Linux users: Rasmol can be launched from a linux cammand line by typing the cammand:
  linux:> rasmol <r>

(Resize the Rasmol window to the desired size.)

Expolring with Rasmol menus:

• File: Open 3dfr.ent

This will open the protein structure in the file 3dfr.ent and display it all in wireframe

• Display: Cartoons
  Colours: Structure
  Options: Specular
  Options: Shadows

This will display in cartoons mode the secondary structure elements that make up the protein
structure.



Rasmol colour for secondary structure
- Helices - Beta strands (beta-sheet) - loops (turn) - unclassified conformation



- How many types of secondary structure elements there is?
- What are they?
- What final level of structure this protein take up?

• File: close

This will close the current display and allow for loading another file

• File: Open 1gzx.ent

This displays the hemoglobin structure

- use what you learned so far to find out what types of secondary elements
is this protein made of. Write down what Rasmol commands did you use.

• Colours: Chain
  Display: Spacefill

- How many complete teriary structure units is hemoglobin made of?
- What do you describe with the level of structure hemoglobin takes up?

• File: close

Scripting with Rasmol:

• File: Open 3dfr.ent
• View: Command Prompt
Display seconday structure in Cartoons and backbone modes:


• Rasmol> wireframe off <r>
  Rasmol> structure <r>
  Rasmol> colour structure <r>
  Rasmol> cartoons <r>

The command Structure gives a summary about the number of Helices, b-Strands and Turns.
take your time to explore the scene.

• Rasmol> cartoons off <r>
  Rasmol> backbone 50 <r>

- The value (50) given to the backbone command is for setting the thinkness of the backbone.
- This will display the molecule in thin line drawn between C-alpha atoms of each residue along the
whole protein creating simplified display called backbone.

Seconday structure display will be clearer.

• Using the mouse to click on C-alpha

The name and number of the picked residue will be displayed in the command line window.

- by mean of mouse clicking, find out the begining & end of each secondary structure element and write them down.

Find Bond distances, Bond-angles and Dihedral (torsion) angles:


- Bond distance: distance between two atoms.
- Bond-angle: angle between three atoms.
- Dihedral (torsion) angle: angle between four atoms.

To do these calculations, we use the Rasmol menu and the Command Prompt
Use the backbone scene, and Zoom In to one of the b-strands.

• Settings: Pick Distance
  Click on two consecutive C-alpha atoms.
  (Distance value, in Angstroms, will be shown in the Command Prompt window)


• Settings: Pick Angle
  Click on three consecutive C-alpha atoms.
  (Bond value will be shown in degrees)


• Settings: Pick Torsion
  Click on four consecutive C-alpha atoms.
  (Torsion value will be shown in degrees)

Display & View Stereo images:


• Rasmol> background white <r>
  Rasmol> Stereo <r>

Stereo mode allows for depth viewing of the scene.

The technique to view stereo pairs of images is based on forcing each eye to see one of
the images, i.e. right eye will see left image and left eye sees the right image. The brain
will merge the two images into one 3D looking images.

In practical terms, you need to gaze into the center of stereo pair and with some little
eyes crossing which will make you see 4 images then try to merger the two closest images.
This with some practice would allow you to see a single 3D image of the sceen.

Tip: While holding your head's position still, try to use the Zoom In and Zoom out function
get the stereo images closer to you or furthest from you.

Display main-chain:


• Rasmol> stereo off <r>
  Rasmol> backbone off <r>
  Rasmol> select mainchain <r>
  Rasmol> colour CPK <r>
  Rasmol> wireframe 50 <r>

  This will display the main-chain atoms along the whole molecule coloured in CPK.
  
  The CPK colour scheme for elements is based on the colours of the popular plastic
  space-filling models developed by Corey, Pauling and Kultun.
  
  The value (50) given to the wireframe is for setting the thikness of the wireframe.

• Rasmol> spacefill 100 <r>

This will display the main-chain atoms in sphirical mode.

Display side-chains:


• Rasmol> wireframe off <r>
  Rasmol> spacefill off <r>
  Rasmol> select sidechain <r>
  Rasmol> colour CPK <r>
  Rasmol> wireframe 50 <r>

  This will display the side-chains found in the whole molecule coloured in CPK.
  (CPK: A popular atoms coloring convention by the chemists Robert Corey and Linus Pauling, and improved by Walter Koltun. )

• Rasmol> colour structure <r>

This will display the side-chains according to seconday elements they belong to.

- add a thin backbone to the scene.

Display water molecules:


• Rasmol> wireframe off <r>
  Rasmol> select water <r>
  Rasmol> colour CPK <r>
  Rasmol> spacefill 50 <r>

  This will display the distribution of water molecules on their own.

• Rasmol> select protein <r>
  Rasmol> wireframe 50 <r>

This will display the water molecules together with the protein.

Display Single Residues, by name and by number:


• Rasmol> select <r>
  Rasmol> wireframe off <r>
  Rasmol> spacefill off <r>
• Rasmol> select protein <r>

  This will clear previous setup and make sure we have protein selected.

Display all Tyr residues and display them in blue:

• Rasmol> backbone 50 <r>
  Rasmol> select Tyr <r>
  Rasmol> wireframe 50 <r>
  Rasmol> colour blue <r>

Display all Glu residues and display them in red:

• Rasmol> select Glu <r>
  Rasmol> wireframe 50 <r>
  Rasmol> colour red <r>

- Comment on what you see in terms of the differences between these two amino acids.

Display residues by residue number, colour in CPK and centre them within the frame of the display:

• Rasmol> backbone off <r>
  Rasmol> wireframe off <r>
  Rasmol> select 20,30,40 <r>
  Rasmol> wireframe 50 <r>
  Rasmol> centre 20,30,40 <r>
  Rasmol> colour cpk <r>



- Display residue 15 (which is Lys) by itself.
- Calculate and record the Bond distances, bond angles and torsions.
- What are the torsion angles called and what are they?

Display a range of residues (only by residue number):


• Rasmol> select <r>
  Rasmol> wireframe off <r>
  Rasmol> select 30-45 <r>
  Rasmol> wireframe 50 <r>
  Rasmol> centre 30-45 <r>
  Rasmol> colour cpk <r>
  Rasmol> cartoons <r>

Note that you can select more than one range of residues by eparating ranges with commas.

- Use what you learned so far to diplay only helices and beta-sheet(s) elements
in cartoons mode coloured by structure. Write the Rasmol commands you used to accomplish the scene.

Display Ligands:


• Rasmol> select <r>
  Rasmol> wireframe off <r>
  Rasmol> cartoons off <r>
  Rasmol> select ligand <r>
  Rasmol> wireframe 50 <r>
  Rasmol> centre ligand <r>

This displays all the ligands bound to the protein.

- Identify the ligands names and residue numbers.
- Display each, in separate scene, inside the protein pocket where they bind.

Display Residues Interaction Environment:


• Rasmol> select <r>
  Rasmol> wireframe off <r>
  Rasmol> cartoons off <r>
  Rasmol> restrict within (5.0,26) <r>
  Rasmol> wireframe 50 <r>
  Rasmol> centre 26 <r>
  Rasmol> select 26 <r>
  Rasmol> spacefill 100 <r>

The command restrict works like select except that it display any atoms found
in a specified redius. The value 5.0 is the radius and the number is residue number
which in this case is for the residue Asp

The commands select 26 and spacefill 100 are optional and has been added
to distinguish the residue in the center of the radius.

- Choose one of the ligands and repeat the above scene for it.
- Try to find as much as you can of possible contacts between the ligand
and the rest in the scene.
- List the interactions (distances) and record their types and say why?