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There are several programs that allow the inspection and manipulation of 3-D structural protein data. In this course we use the swiss protein data bank viewer, aka Deep View. 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).
SPDBV is a good choice, because it also provides an interface to the Swiss Protein databank modeling software.
The SPDBV program is available free of charge from the Swiss Institute of Bioinformatics.
There are several on-line tutorials available to learn the use spdbv:
The exercise in this section is taken with slight modifications from Gale Rhodes' basic tutorial, many of the exercises in the following sections parallel exercises in the basic tutorial.
You can retrieve pdb files from the NCBI, or from the protein structure data bank at Rutgers University. (To do so search for the file -use only the name, omit the extension-, once the page for the structure has loaded, click the download link in the upper right hand corner and click on the link that indicates to download the uncompressed pdb file.) (The ones used in the course are also available here - we will use 1HEW.pdb and 1bmf.pdb today)).
Create a folder on the desktop (in finder - the application that runs when you click somewhere on the desktop- select file, select new folder). Name the folder with your name.
Save SPDBV_4.1.0_OSX.zip to this folder (ctrl click, select download linked file as ...).
Double-click the zip file to extract the folder. Inside the folder, you will find the "Swiss-PdbViewer" application. Drag the icon down to your dock, so that you will be able to find it again easily.
(Here is the original download location, in case you are using something other than a Mac.)
MORE PREPARATION
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Do the following:
copy the files labeled 1bmf and 1HEW from here into a folder on the dektop of your computer (go to the listing, ctrl click on the name and "save link as ..." to a folder on your computer. <ctrl click> means that you press down the contol key on the keyboard at the same time as you click with the mouse. In a two button mouse, this corresponds to a right click. One suitable folder to copy things to is the folder called download in the SPDBV folder).
Double click on the icon "Swiss-PdbViewer" to start SPDBV.
load 1HEW.pdb (At the very top on the screen, click on file, then open PDB file).
There is the toolbar panel and located above the window with the structure, but below the program bar at the top of the screen. It looks like this:
The button which is second from the left allows you to move the structure in the structure window. Click on it and then click and hold in the structure window to drag the protein to the desired location. The third button from the left allows you to zoom in or out. Click on it and then click and hold in the structure window to drag the protein larger or smaller. The fourth button from the left is the rotate button. Click on it and then click and hold on the structure window to rotate the view.
The leftmost button centers the structure in the structure window. Click it now to return the molecule to the center of your screen.
Explore Different Display Options. For large structures, render in 3D may slow down the program, but for small ones like 1HEW, render in solid 3D is a useful choice.
click on the page icon (in the second row of the tool bar and is a white rectangle-- you may need to expand the menu window to see the page icon) and scan through the pdb file.
The Control Panel looks like this:
open the control panel (in the WIND-menu at the very top of the screen).
open the alignment window (in the WIND-menu at the very top of the screen)
select all (to do this, either hold down the Apple key and press the letter A (Apple-A) or look under the select menu at the very top of the screen).
in the WIND-menu, click Ramachandran plot
in the control panel, select different residues (click on the 'h' and 's' in the first column, then hit return (return make the selected residues visible, else the visibility and the selected residues can be different!). How does the display change in the Ramachandran plot? In the main window? (For more info on the Ramachandran plot see http://www.bmb.uga.edu/wampler/tutorial/prot2.html)
select all, hit return
Explore different coloring using the color menu at the very top of the screen (CPK, secondary structure, accessibility) and display options in the display menu at the top of the screen (show CA trace only, show oxygen, ...) Often the amount of information is overwhelming.
REMARK: If you do serious work save your work periodically, sometimes it is impossible to recover from inadvertent mouse clicks). If you used the interactive feature of the Ramachandran plot to (inadvertantly) modify the structure, re-load the original 1HEW file.
Remark2: There is a difference between select (the residue turns red in the control panel) and actually seeing the residue in the main window. If you hit return the selected residues become visible.
Remark3: Especially when working with large structures, it is beneficial to only display the alpha carbons of the backbone. To do so, shift mouse click in the side (side chain) column of the control panel (to turn on/off all checkmarks in this column). In the display menu uncheck "show backbone oxygens", and check "show backbone as alpha carbon trace".
Remark4: Another way to have an overview picture is to select ribbon representation in the control panel. Uncheck (shift mouse click) all other columns and check the rbn (ribbon) column. To have color commands act on the ribbon display, you need to select the ribbon as target for the color commands (either via the first item in the color menu, or via the little black triangle below the "col" (color) header in the control panel.
To highlight residues in the substrate binding pocket do the following: Select (point the cursor over the NAG201...at the buttom of the control panel) the NAG inhibitor (shift click adds to the selection).
Color CPK
Invert selection (in the SELECT menu at the top of the screen)
Color secondary structure
Invert selection
In the Tools menu at the top of the screen, choose compute H-Bonds
click "side -" column in the control panel to turn the sidechain display off
Select only the NAG inhibitor in the control panel
In the select menu at the top of the screen, select Neighbors of selected aa - check select add to selection button
hit return
click on the + under the "side" header in control panel (acts only on selected residues)
In the select menu at the top of the screen, select group properties, and then Non-polar aa
click on Header COL in Control panel select a blue color to color hydrophobic residues blue
Are there "blue" residues interacting with the N-Acetyl glucosamines? How come?
Can you locate which one of the Tryptophan residues "sits under" the second of the N-Acetyl glucosamines?
Play around, if in doubt use the ? buttom.
The worst that can happen is that you'll have to restart your computer. SPDBV allows you to model protein structures, i.e. you can actually change the structure (e.g., you can drag a dot in the Ramachandran plot; this changes the structure irrevocably). If this happens to you by accident, close the structure and reload. If you work on something important save you work frequently.
Open the alignment window and display the complete lysozyme molecule. Observe the color change in the structure that happens when you move the mouse over the sequence in the alignment window.
The resulting display after some beautifications might look like this:
yellow: the NAG inhibitor; blue: residues in the binding pocket that are non-polar, depicted as space filling balls; red: other amino acids in the binding pocket; gray: the rest of the Lysozyme molecule, but only the backbone.
Trouble shooting: In case your cartoon (ribbon) display does not look nice: 1st: in the Control panel window, check that the coloring commands are selected to pertain to the the ribbon. 2nd: under preferences, select ribbon, and place a check mark in the field "render as solid ribbon"
Other things to try: 3D rendition (in the display menu), slab view (shift and mouse forward/backward move the slab through the molecule, shift and mouse left/right change the slab size), explore the make up of the pdp file (text icon below the cursor control).
If you <alt> click on a residue in either the alignment window or the control window, the display centers on this residue.
shift and mouse click adds residues to the list of selected residues (works in either window)
Can you obtain a figure similar to the one below?
Go to the control panel click on the little black triangle to the right of the col column and select color ribbon, then secondary structure in the color menu. Display ribbon in the control panel, remove the other displays .....
Exercise 2:
Aligning F-ATPase alpha and beta subunits
Start SPdbV
Open 1bmf.pdb
Color Chain
Change color chainD to grey/blue (left click in control panel on D in first column to select chain D, right click on COL, select color)
Scroll down the control panel and select all ATP analogs (labelled as ANP, ADP or ATP)(press ctrl key and right click to select)
right click on COL in heading and select red color
Read the pdb file to get info on which chain is which
select chain F (including nuc) and save selected residues as betaTP.
select chain A (including nuc) and save selected residues as alphaE.
After playing with the F1-ATPase, close this file and open betaTP and alphaE.
In WINDOW - Display layer info
select and display only the nucleotides (ANP600)
There are different ways to align 3-D structures. One way is to select 3 corresponding points in each of the two structures. To do so you can use the substrate molecule.
Using the mov check off in the Layer Info, reorient the two AMPs so that they are in a similar orientation (but not overlapping).
Click on the align bottom with the 3 green and 3 red dots. Notice the red instructions that appear in the header next to the pdb-page icon. Follow these instruction using three corresponding atoms.
SHIFT DISPLAY CA chain (Shift makes the commands act on both layers)
Using the mov checks in the Layer info, move the two chains next to each other.
What do you think about the result?
Another way to align structures is to use the magic fit in the tools command. Do this and run improve fit (notice the red info in the header)
Click on alpha in Layer info to make the alpha subunit the active layer
Make the beta subunit the active layer
COLOR rms . The further the atoms in the beta subunit are away from the alpha subunit, the longer wavelengths it is the colored.
WINDOW display alignment window - gives you the aligned sequences.
Which part of the molecule looks different between the Alpha vs. the Beta subunit?
Is the Walker motif (G--G--GKT) well aligned in the structures ?
Choose one of the following options, then click the button.
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