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Objectives for today: 

• Launch chimera
• Display a 3 D coordinate file from the pdb (1HEW) in chimera
• Use differnet display settings
• Display amino acid side chains in the binding pocket of 1HEW and study the interactions between the substrate and the binding pocket.
• Calculate a Ramachandran plot, and determine where in this plot alpha helices, beta sheets, and glycine residues fall. 
• Save your work as image and project. 

Introduction
(This is background information for you to read and explore. You do not need to follow any of the links listed in the introduction, but these might come in handy, if you want to install software on your own computer.)

We will use the chimera program to visualize and analyze protein (and other molecular) structures. If you use your own computer, the program is available for different platforms at http://www.cgl.ucsf.edu/chimera/download.html  
Notes: In previous years we used the Swiss protein databank viewer (SPDBV), but the updates for the program lack behind the changes in the operating systems.
An alternative, very popular to generate rotating or rocking images is pymol . A very simple get to know pymol exercise is here.  (If you think protein structures are in your future, you might want to give this a try in your own time.  For many of the more difficult things there are pretty useful YouTube tutorials).

You can retrieve pdb files from the NCBI, or from the protein structure data bank at Rutgers University. But if you know the name of the protein data bank file (extension pdb) you can use chimera to download the file. (The ones used in the course are also available here - we will use 1HEW.pdb and 1bmf.pdb today).

Preparation

For the instructors to get to know you, and to allow a more organized way to help students, please write your name on a small piece of paper (or on an the wings of an origami crane). If you have a question and the instructor is busy helping other students, please move your name card onto the top of your screen, the instructors will try to help you ASAP.

Exercise 1 :

Do the following:

Log into the computer using your netID.

Find the chimera program on the PC start the program through double clicking the icon.  Chimera is a program to visualize and analyze protein (and other molecular) structures. 

If you manage to obtain a beautiful display, save the image as a jpg image and save the session (from the file menu) and send me a copy per email.

We will use the structure for lysozyme crystalized with an inhibitor, a trimer of N-acetyl glucosamine.  The normal substrate for lysozyme is cleave the sugar backbone on the cell wall of bacteria.  This murein sacculus surrounds the bacterial cell like a chain link armor, and is creates the cells turgor pressure in response to the osmotically driven water influx.  When the sugar backbone is cleaved, the elasticity of the cell wall decreases, and the cells explode due to the osmotically driven water influx.   In the normal back bone of the cell wall, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) are alternating and linked together through a beta 1->4 bond (the same type as in cellulose).  The lactic acid side chain in NAM is used to cross link the sugar polymers through short peptides.  The lactic acid side chain is missing in NAG.  The inhibitor (NAG)3 is bound to lysozyme, instead of (NAG-NAM)n, but is is not hydrolyzed, allowing to study the interactions between the substrate and the binding pocket of the enzyme. 

• In the chimera program under the file menu, select fetch by ID, place a checkmark in pdb (denoting the protein databank format) and enter 1HEW.  You should see the structure in a ribbon representation, and the inhibitor.  You also see some of the sidechains that are part of the binding pocket. 
• Explore the different ways to move the structure with the mouse (click and move, right click and move, option click and move, command click and move). 
• Which key/mouse combination allows to zoom into the structure, and which allows to move the structure sideways or up and down without turning it?

• In the presets menu, explore the different interactive displays.  Return to the ribbon display. 

• Open the viewing controls in the tool menu and select side view.  This interactive window allows to relocate the viewpoint and to view only a slab through the structure (you can move the yellow lines, and will see on the slab between the two planes (represented by the two lines).  (if you view the molecular surface, it might be a good idea to cap the surface of the slab in an unusual color, so that you recognize it as a surface created through the cut). 
  The tabs in above the viewing menu also allows you to change the lights and the camera. For example, you could choose a stereo view, but to see this in 3D you need to be good in seeing cross-eyed - if you want to try it out, switch the camera to cross-eyed stereo, hold a pen or you thumb halfway between the screen and your eye, focus on the pen. If this works, you see three versions of the structure, the one in the middle is in 3D. It might help to adjust the distance between your eyes, and the distance to the screen). Return the camera to normal. Be careful, I did not bring aspirin.
  
  
• To highlight and color secondary structure elements: 
  Select > secondary structures > helix then
  Actions > color > red  (NOTE: actions apply only to the items selected!)
  Select > secondary structures > strand then
  Actions > color > yellow
  You can leave the coils as they are, or color them in a color of your choice. 
• (Alternatively, you can go to Tools > Depiction > color secondary structure and then select colors for the different secondary structural elements.)  

Next we will try to study the interactions between the NAG trimer and the binding pocket.  In particular we are interested in the possibility of hydrophobic interactions between the substrate and the sugars.  There are, as usual, many different ways that lead to similar results.  Share the results with your neighbor!

• Possibility 1:
  select the NAG trimer
  Select > Residue > NAG
  Select > Zone > place check mark into “angstroms from currently selected atoms , DO not place a check mark into “ Select all atoms …. “
  Actions > Atoms/Bonds > sidechain/base > show
  Then Actions > surface > show
  (you might need to adjust the coloring.)
  Then select the NAG trimer again and hide the surface view. 
  (select > residue > NAG then Actions surface hide).
  The result should display the sidechains of the binding pocket as space filling models, with the substrate as wire diagram.  Can you see the interactions between a tryptophan and the sugar?  Which tryptophan interacts with the central NAG?  (Actions > label > name and specifier)
  
  
• Possibility 2:  select the NAG trimer
  Select > Residue > NAG
  Actions > surface > show
  Actions > color > by element
  Select > Zone > place check mark into “angstroms from currently selected atoms , DO not place a check mark into “ Select all atoms …. “
  Display the side chains of the binding pocket: Actions > Atoms/Bonds > sidechain/base > show
  Can you see the interactions between a tryptophan and the sugar?  Which tryptophan interacts with the central NAG?  (Actions > label > name and specifier)

• Possibility 3: 
  choose Preset > Interactive 3, to display the hydrophobicity surface. 
  Select > Residue > NAG
  Actions > surface > hide
  If you only want to see the binding pocket.
  Select > Zone > place check mark into “angstroms from currently selected atoms , DO not place a check mark into “ Select all atoms …. “
  Select > invert
  Actions > surface > hide
  Can you see the interactions between a tryptophan and the sugar?  Which tryptophan interacts with the central NAG?  (Actions > label > name and specifier)

In a few words describe the hydrophobic interactions between the substrate and the enzyme that you see.

Which tryptophan interacts with the central NAG?

• Obviously. electrostatic interactions sand hydrogen bonds play a role. 
  To see the hydrogen bonds, Tools > Structure Analysis > find H Bonds
  If you apply this to the ribbon structure, you see the many H bonds that stabilize the secondary structural elements. 

Here is the lysozyme with the colomb surface colored:

Calculate and Draw a Ramachandran Plot

The chimera program comes with a command line. 
To see the command line select Tools > General controls > Command line.  
[A command line reference is in the chimera user guide (>Help > User’s Guide) on a mac, this is here. Some commands can also be executed through the model panel (General controls > model panel).]
Open the command line window, type ramachandran <return>
To explore where in the Ramachandran plot different secondary structure elements fall, select the different structural elements (alpha helix, beta sheet (strands), coil), and observe how the color of the selected amino acids changes in the Ramachandran plot. 
Then Select > Residue > Gly. 
Why do glycine residues in the Ramachandran plot often fall outside the areas occupied by the other amino acids? 

Ramachandran plot for 1HEW with glycine residues in red.

If you have time, do the following, we will return to this next week!

ATPase subunits

The ATP synthase (aka as proton pumping ATPase) consists of ring of proteolipids that are integrated into the membrane, a head group (which is the structure in 1bmf), and a stator that keep the non-rotating parts fixed.  The head group known as F1 portion of 6 ATP binding subunits (3 alpha and 3 beta subunits).  The beta subunits bind and hydrolyze ATP, if the enzyme works as a proton pumping ATPase.  These catalytic subunits rotate the central gamma subunit.  In the intact enzyme, the gamma subunit is linked to the proteolipids, which than rotate relative to the stator.  When they pass the stator the proteolipids (proteins that behave like a lipid, but they do NOT contain a lipid) they undergo a motion that moves a glutamate or aspartate residue into a different environment, where is picks up or dissociated a proton.
How is ATP synthesis coupled to the electron transport chain? 

Why is the ATPsynthase important?  Make a guess as to how much ATP are synthesized in the human body per day. 

The beta and alpha subunit evolved from a very ancient gene duplication (this duplication had already occurred in the common ancestor of Bacteria, Archaea, and the eukaryotic nucleocytoplasm); this duplication had already occurred over 3.5 billion years ago.  This means that the two subunit types (alpha and beta) evolved as separate subunits for over 7 billion years.   

1) Open chimera and open the 1bmf file (File > fetch by ID 1bmf)
Look at the structure in the first two preset modes (the surface may take some time to compute).  Note the central gamma subunit (consisting mainly of alpha helices). 
[aside: in the related structure of a transcription termination factor, which unwinds a newly synthesized mRNA from the DNA template, the six ATP binding subunits have a similar arrangement and the place of the gamma subunit is taken by the RNA DNA duplex].  Also color the Ribbon by secondary structure (Tools > depiction > secondary structure).
Select all non-standard residues (select > residue > ...) and show them as ball (Actions > Surface >show).  Can you determine which chain, via (Select > chain), does not have an ATP or ATP analog bound? 

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