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Follow up from last computer lab on protein structure exercises

All the .PDB files can be found here.

Part 1 : 30 minutes

Aligning F-ATPase alpha and beta subunits

Start SPdbV

Open 1bmf.pdb

Color Chain

Change color chainD to grey/blue (rightclick 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

Color CPK

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 2 : 20 minutes

Exploring intein structures in SPDBV

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 .

• Saccharomyces cerevisiae intein (PMID: 9160747, 1VDE ),
• the same Saccharomyces cerevisiae intein bound to its recognition sequence (PMID: 12219083, 1LWS ),
• the Mycobacterium xenopi mini intein (PMID: 9437427, 1AM2 )

1. Open 1VDE in SPDBV. This structure has two chains. Save chain A to a separate file.
   
   
2. Reopen chain A in SPDBV. Open Mycobacterium mini intein 1AM2. Align two structures using Magic Fit. How good is the alignment? (your answer should be "Bad").
   
   Depict the structures as ribbons and color them according to the secondary structure. Rotate two structures until you can see the similarities between mini intein and one of the domains of large intein. Move them on top of each other. Do "Fit -> Improved Fit...". Does it work now?
   
   Can you find which part of Saccharomyces cerevisiae intein corresponds to the endonuclease domain by comparison of the two structures? [Manually inspect the two structures to find the similarities]. Color the putative self-splicing and endonuclease domains of 1VDE in two different colors. Select N and C terminals (First a.a. and the last a.a.) in both structures. How close are they (in angstroms)? [Click on the button with "1.5A" label, and select first and second a.a. to obtain the distance]. Save your project.
   
   
3. Open Saccharomyces cerevisiae intein that is bound to its recognition DNA sequence. Display intein (chain A) as ribbons with secondary structure color scheme (select color target with little black triangle in Control Panel). Color DNA as CPK, and compute hydrogen bonds (Tools - > compute H-bonds). Does the finding of DNA interaction domain agree with your previous assignment of the self-splicing domain? (see the saved structure from the previous exercise)
   
   
4. Try to find a way to display the interactions between the aminoacid side chains and the DNA helix. One way to do it is to select two DNA chains and select Neighbors of selected amino acids. Choose "select groups that are within" option. Click on the "Cloud" icon in the header of the control panel to display the aminoacids as balls. Also click in the header for showing sidechain. Manually turn off the cloud checkmarks for the DNA. One way to look at individual interactions is to turn the molecule so that one looks down the DNA helix, and select the "Display -> Slab" option. If you press shift while mouse cursor up and down the visible slice moves through the molecule along the axes perpendicular to the screen. If you press the shift key and move the mouse left and right you increase or decrease the size of the slab.
   
   
5. The Lys 340 and Glu 366 are residues that are important for interaction with DNA. Select those residues (in Control panel choose label column to depict the label). Do they interact with major or minor groove of DNA? Which base pairs interact with these amino acids?
   

Get a headstart on assignment #7

If you have time you could start on take home assignment #7:
Align the the two sequences datasets using clustalx, do a bootstrap analysis
in clustalx (WRITE DOWN WHICH PARAMETER SETTINGS YOU CHOSE!).
Save the alignments in phylip format into the PHYLIP folder.
You could use the following programs in sequences:
SEQBOOT (use only 100 samples),
PROTDIST or DNADIST (depends on which of the datasets you choose, multiple
datasets, among site rate variation?),
FITCH (multiple datasets),
CONSENSE
or SEQBOOT, PROTPARS/DNAPARS, CONSENSE

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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