MCB 5472 : protein structures in pymol

You should answer the questions in red!

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 use this in your student project.)

There are several programs that allow the inspection and manipulation of 3-D structural protein data. In the past we used the swiss protein data bank viewer, aka Deep View or SPDBV.

While this is an excellent choice, in part becasue it also provides an interface to the Swiss Protein databank modeling software, the program pymol has generated a large following, and it can be more easily adapted to do interesting things.

The pymol program is available free of charge for educational purposes. You can down load executables from the pymol site (check with the instructor for the password), or you can get the latest version form sourceforge, older versions are also available through different servers (see here for a windows example), More information on how to install pmol is available in th pymok-wiki

There are several excellent on-line tutorials available to learn the use pymol, the most authorative is part of the pymol wiki, but there are dozens of you-tube clips on how to do things in pymol (a discussion on how to align structures is here)

Task 1: Install pymol on your computer

Start pymol either through typing pymol in the terminal window (in case you downloaded the sourceforge version for unix) or by double cliccking on the Pymol or MacPyMOL icon.

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.) (My favorite ones are also available here).

1HEW - getting to know pymol part 1

To get to know pymol, type fetch 1hew in the pymol command line. The text window should display information on the file, the graphics window should display the structure. (You also can download the file 1hew.pdb from the pdb databank, and then use the file menu to open the file. To familiarize yourself with pdb files, go to the pdb databank, search fro 1HEW and display the pdb file.)

What molecule is depicted in 1hew ?

To the right of the graphics window are four buttons

• A - Actions: Rename, duplicate, remove, apply presets (like "ball-and-stick" or "publication"), perform computations
• S - Show: Change the way things appear, e.g. change to stick or cartoon view.
• H - Hide: Things that are shown using S accumulate, and don't automatically replace the last view. H is the opposite of S and hides unwanted representations.
• L - Label: Label atoms, residues, etc.
• C - Color: Change the color of atoms and groups.

The bottom right gives the settings for the mouse, you can select the mouse that you use in the mouse menu on top.

For the 1HEW structure, H everything, then S cartoon, then C by chain -> chainbow.

Explore different display options, view and cursor options.

At the bottom right is the Select button. click it, this displays the sequence of the lysozyme. At the end of the amino acids are the non amino acid atoms that are part of the structure. Select the N-Acetyl Glucosamine (NAG) trimer. This opens a new row of ASHLC buttons. select S sticks and C one of the chnops coloring schemes.

If you get lost, right-click the background of the viewer, and select reset from the Main Pop-Up. The model should return to view.

Try to depict the protein as a meshed surface and the substrate as space filling balls. (Challenge: Can you find a way to depict the surface according to charge? What commands did you use?)

To make a simple movie select movie in the main menu, then ->Program->Camera->Y-Roll-> 32 Seconds
then press the play button on the lower right. Unselect loop frame, add some other movie parts such as nutate, or rock.

Getting to know pymol part 2

Repeat the above exercise with four favorite molecule.

• Download your favorite crystal structure from the protein databank. Be sure to download the .pdb file
• Load sequence into pymol: File > Open (alternatively, you can use the fetch command as in the last exercise)
• To see the sequence along with the click Display -> Sequence on
  What do the different sequence modes do?
• Explore different coloring and display options. (Any intersting observations?)

To create a high resolution movie using ray tracing

• Enter the following commands into the PyMOL> bar above the protein followed by enter
  • mset 1,180
  • util.mroll 1, 180, 1
  • set ray_trace_frames, 1
  • set cache_frames, 0
• Then save as a movie
  • File>save movie as>Quicktime
  • The frames per second is how fast the protein will rotate the higher the number the faster.
    Try 12, 25 or 60. Which seems to be a speed that works well?

a.    We will color a protein according to its conservation profile and determine where the inteins sit in the VMA protein

b.    You will need the conservation profile and protein .pdb file

c.     Lets work with the excel file first (note: a perlscript that creates a conservation profile from a multiple sequence alignment is here)

                                               i.     In this file the first column gives the alignment position and the second column is the conservation score - the lower the more conserved (the number of aa types in a sliding window)

                                             ii.     Sort the profile on the second column smallest to largest

                                            iii.     We’ll assign colors in the third column for now working with a ROYGB color scheme

1.     Label 0-2 Red

2.     Label 2-5 Orange

3.     Label 5-8 Yellow

4.     Label 8-11 Green

5.     Label 11 – the rest Blue

                                           iv.     Save your file and put it a side for now

d.    Now will work with the protein files

                                               i.     Open 1VDZ.pdb in PyMOL

1.     On the right under the all object click H everyting, then S ribbons

2.     In the PyMOL> bar type

a.    color gray, all

3.     We are going to make each color group an object. This will be helpful if we want to go back and change the colors for the group later.

4.     For each color we marked off in the excel file select the amino acids from the first column

a.    So for the reds select those copy them then in another cell right click > paste special and select transpose

b.    Copy the transposed list and paste it in text wrangler

c.     Then copy the space between the numbers

d.    Then Search>find

e.    Paste into the top box and replace all with what between, the quotes here ‘ resi ‘ so space resi space

f.      Back to pymol

                                                                                                     i.     Type select RED, resi then copy and paste the line from text wrangler into the PyMOL> box

                                                                                                  iii.     Now all those amino acids are grouped together

                                                                                                 iv.     Do the same for the rest of the colors replacing the object identifier “RED” with the appropriate color name.

g.    Once all the groups are made they should show up on the side bar

h.    Make one more group for the inteins

                                                                                                     i.     select intein insertion site, select intein_site, resi 249 resi 269

i.      Color each group

                                                                                                     i.     color red, RED

                                                                                                   ii.     color yellow, YELLOW

                                                                                                  iii.     ect.

j.      To make the intein insertion sites pop out against the rest of the protein select intein on the side bar click S >as spheres

5.     Where are the inteins sitting?

Other intersting structures:
small intein without homing endonuclease (HE) from Mycobacterium xenopi mini intein (PMID: 9437427, 1AM2)
intein with HE from vacuolar ATPase in yeast (PMID: 9160747, 1VDE)
intein with HE from yeast bound to DNA (PMID: 12219083, 1LWS)

Histone core (8 histones) with DNA wound around it (1AOI)

ATP synthase head group (F1) from beef heart mitochondria (1bmf)