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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.
SPDBV is an excellent choice, because it also provides an interface to the Swiss Protein databank modeling software.
The SPDBV program is available free of charge at the expasy homepage.
There are several excellent on-line tutorials available to learn the use spdbv:
A basic tutorial is at
http://www.usm.maine.edu/~rhodes/SPVTut/index.html ,
And a course on structure, spdbv, and modeling is at
http://www.expasy.ch/swissmod/course/course-index.htm
The exercise in this section is taken with slight modifications from Gale Rhode's the 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, click the explore link and right click on the link that indicated download uncompressed pdb file.) (The ones used in the course are also available here ).
Do the following:
copy all files listed here onto your computer (go to the listing, ctrl click on the name and save to a folder on your computer).
Locate the folder that contains the program SPDBV.
Start SPDBV through double clicking on the icon
load 1HEW.pdb
click on the three cursor control buttons and rotate/move/enlarge the picture of the lysozyme molecule
click on the center molecule button to the left of the cursor buttons
click on the page icon (you may need to expand the menu window to see the page icon) and scan through the pdb file
open the control panel (in the WIND-menu).
open the alignment window (in the WIND-menu)
select all
in the WIND-menu, display 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)
Explore different coloring (CPK, secondary structure, accessibilty) and display options (show CA trace only, show oxygen, ...)
REMARK: If you do serious work save your work periodically, sometimes it is impossible to recover from inadvertent mouse clicks)
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.
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)
Color secondary structure
Invert selection
Tools compute H-Bonds
alt click "side" column in the control panel to turn the sidechain display off
Select only the NAG inhibitor
select Neighbors of selected aa - check select add to selection buton
hit return
click on the "side" header in control panel (acts only on selected residues)
select group properties Non-polar aa
click on Header COL in display 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 Tryptophane 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.
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), have a look at the opening control window (upper left icon below the cursor control).
If you right 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: 20 minutes
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 ANP analogs (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 structure base amino-acids alignments?
Part 3 : 20 minutes
Background information: Most inteins are composed of two domains: one is responsible for protein splicing, and the other has endonuclease activity. A few inteins have lost the endonuclease domain completely and retain only the self-splicing domain and activity. The latter inteins are called mini-inteins .
Optional exercises #1 :
Comparing divergent proteins with similar structures
load 2DLN.pdb load 1GSA.pdb Are these structures similar? homologous?
Does Magic fit work?
For both structures Display the CA backbone only and color according to secondary structure. Use the layer info panel and orient the two structures so that they look similar.
Select the ADP molecules only
Use the 3 point alignment approach to align the ADP molecules
make the whole molecules visible again
move one structure over to the right (no turning)
select the ADP molecules
In the control panel header click on the cloud icon to display both ADPs in space filling mode.
In display click on render Q3D.
(To get more spectacular displays, you can save the pictures as POV files and use the program POV ray to make even nicer images)
If you have time, do the same for 1GSA, 2DLN and CPSBfrag and CPSFfrag . The latter two files are clippings of the front and back ATP binding sites of the carbamoyl phosphate synthetase (1BXR).
WHAT DOES THIS MEAN? Recall the use of 2DLN in PSI blast. Are all of these structures homologues? What does that tell you about evolution of proteins? An illustration is here .
Optional exercises #2 :
If you have more time to spare and you are up for a challenge, take a look at the nucleosome. Right click here and save as pdb file. Open it from within spdbv. You might want to do some of the future exercises with the nucleosome in addition to the ATPases - thus save the pdb file, where you can find it again.
Align all the histones form the nucleosome to one reference histone and color in rmv:
The result might look something like this:
The picture shows a structure alignment of the 8 histones (2 each) that are part of the nucleosome. All the histones were colored regarding the match to H2A, except H2A, which was colored according to its match to H3. Coloring option RMS - shorter wavelengths - better match
Below same as last figure, but histones are depicted side by side :
Below are two views of the complete nucleosome. Histones H2A are depicted as spacefilling balls and RMS colored regarding their match to H3. The rest of the molecule is colored according to chain.
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