In-class PyMOL Excercise by liwenting

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									BIOM 200 Module 4 –In-class exercise: PyMOL molecular visualization
PyMOL Educational download – http://pymol.org/ep         How to find structures? Know your four-letter code, then…
   username:      dec2010                                    RCSB Protein Data Bank – http://www.pdb.org
   password:      mol2View (case-sensitive)                  Electron Density Server – http://eds.bmc.uu.se/eds/


More help using PyMOL
User’s Guide (out of date): http://pymol.sourceforge.net/newman/userman.pdf
User’s Guide (out of date): http://pymol.sourceforge.net/newman/user/toc.html
PyMOL Wiki: http://www.pymolwiki.org/index.php/Main_Page
Quick reference sheet: http://www.pymolwiki.org/images/2/20/Refcard.png


Instructions for in-class exercise:

Download PyMOL (Windows or Mac – see above for download instructions)
Download Mad2_excercise.zip from class web site, extract enclosed files.

Load and view structure of peptide-bound Mad2

         File > Open > 1KLQ.pdb
         On 1KLQ line at right: S (Show) > as > cartoon
                                C (Color) > spectrum > rainbow
              This shows the protein as a gradient from blue (N-terminus) to red (C-terminus)
              This can also effectively be done with: A (action) > preset > publication

Explore the Mad2-peptide interface

         On command line:         create peptide, chain B
                                  hide cartoon, chain B
                                  show sticks, peptide
         On peptide line:         C (Color) > by element > [pick one]
         On command line:         hide sticks, elem H
              Now the peptide is shown with covalent bonds as sticks between atoms, and atom types color-coded

         On command line: create Mad2, chain A
         Turn off display of 1KLQ object clicking on its name
         On command line: hide cartoon, Mad2 and resi 151:176
                               show sticks, Mad2 and resi 150:177
                               hide sticks, elem H (get rid of hydrogens for clarity)
         On Mad2 line:         C (Color) > by element > [pick one]
         On 1KLQ line:         A (action) > find > polar contacts > within selection
         On command line: show lines
               Now Mad2 is shown with atom types color-coded, and the area surrounding the bound peptide is shown as
               sticks as well. Hydrogen bonds are shown as yellow dotted lines.

     Explore the molecular interactions between Mad2 and the peptide:
         Use the “Measurement ” tool in the Wizard menu to measure the distance between atoms.
         Can you identify any hydrogen bonds? Any distinctive patterns of hydrogen bonds?
         Can you identify Van der Waals interactions between hydrophobic residues?
         Can you identify any pi-stacking interactions?



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BIOM 200 Module 4 –In-class exercise: PyMOL molecular visualization

        How sequence-specific do you think this interaction might be? Would Mad2 bind just any peptide?
    Try displaying Mad2 and peptide as spheres, mesh, or surface. Examine the shape complementarity between them.
        S (Show) > as > [spheres/surface/mesh]

Compare Mad2 open and closed conformations
      File > reinitialize
      File > Open > 2V64_A_B_E.pdb
      On 2V64_A_B_E line:     S (Show) > as > cartoon
                              C (Color) > by chain > by chain

             You should see three chains: two copies of Mad2, with one bound to a peptide. The peptide-bound Mad2 is in
             the “closed” state, and the unliganded Mad2 is in the “open” state. Next we will overlay them to see the different
             states directly.

        On command line:          create closedMad2, chain A or chain B
                                  color white, chain A
                                  color purple, chain B
                                  create openMad2, chain E

        On openMad2 line: C (color) > spectrum > rainbow
        On 2V64_A_B_E line:  toggle object display off by clicking name

        On command line:          align openMad2, closedMad2
                                  color gray, closed Mad2 and chain A and resi 166:204

             In the open state, the C-terminal two beta strands (orange and red) are located at one side of the beta sheet. In
             the peptide-bound closed state, these two strands (now gray) are displaced by the peptide, and they move to
             the opposite side of the beta sheet. Along the way, they displace the N-terminal beta strand (blue).

             NOTE: the “align” command will work with identical-sequence or similar-sequence objects. Often it will work
             better if you add certain selection criteria, e.g.:

             align openMad2 and resi 50:150 and name ca, closedMad2 and chain A and resi 50:150
             and name ca

             After the align command has refined its alignment, it outputs the route-mean-square (r.m.s.) deviation of the
             atom pairs it has aligned, and how many atom pairs it used in the alignment




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BIOM 200 Module 4 –In-class exercise: PyMOL molecular visualization

Look at electron density for Mad1 in the Mad1-Mad2 complex

        File > Reinitialize
        File > open > 1GO4.pdb
        On 1GO4 line:        A (action) > preset > publication
        On command line: hide cartoon, chain H
                             show sticks, chain H
        Right-click on an atom in chain H: chain > color > by element > [pick one]
        Center on the region of chain H (Mad1) that is bound to a Mad2 molecule like the peptide
        we’ve already seen.

        File > open > 1go4_map.ccp4
        On 1go4_map line: A (action) > mesh> @ level 1.0
           Now you have displayed the electron density at I/sigma of 1.0 (normal view). This is the actual data of the
           experiment. The model is the authors’ best interpretation of this data.

        On 1go4_mesh line: toggle object display off by clicking name
        On command line: isomesh Mad1mesh, 1go4_map, 1.0, chain H, carve=1.8

           You have displayed the electron density at I/sigma of 1.0 (normal view for electron density maps) only within
           1.8 Å of chain H, one of the copies of Mad1. How does the model look compared to the data? Are there any parts
           of the model not represented by the data? Which parts of the model are more likely to not be seen in the map?
           Why?

        On command line:        isomesh Mad1mesh_lo, 1go4_map, 0.6, chain H, carve=3.0
                                color green, Mad1mesh_lo

           Now you have displayed the electron density at I/sigma of 0.6 within 5.0 Å of chain H. Can you see the missing
           parts of the model better?

        Find Arginine 556 of chain H:           In sequence view, find chain H, residue 556.
                                                Right-click on the ‘R’ > center

           Look at the electron density surrounding the side chain of Arginine 556. Do you think this was built correctly? Is
           there an alternate interpretation of this side-chain’s rotamer? What does this tell us about the model? What
           does this illustrate about believing any model from a crystal structure?




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BIOM 200 Module 4 –In-class exercise: PyMOL molecular visualization

PyMOL Reference Sheet




To start your session over: File > Reinitialize

Mouse actions (see Mouse Key above)
                      Drag                     Click
    Left               Rotate view             Select Residue
    Center             Move view               Center view
    Right              Zoom                    Contextual menu
    Scroll             Slab (depth of view)

To save an image: File > Save Image as > PNG…
    To make it look nice, type Ray in the command line first, then save the ray-traced image as above.




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BIOM 200 Module 4 –In-class exercise: PyMOL molecular visualization

Selection Criteria: Use logical operators in series (or nested with parentheses) to select atoms. e.g.:

    closedMad2 and chain A and resi 50:150 and name ca
      selects atoms that fit all the following criteria: in object closedMad2, are in chain A, have
      residue numbers 50-150 (inclusive), and have the atom name ca (carbon alpha).

    chain A or chain B and resi 1:100 and resn Leu (and name ca, cb, o, n)
      selects atoms that are in either chain A or chain B, and have residue numbers 1-100 (inclusive),
      and have the residue name Leu (leucine) and have any of the atom names ca (carbon alpha), cb
      (carbon beta), n (main-chain nitrogen) or o (main-chain oxygen)

    Selector
    [objectname]           Atoms that are in a certain object
    chain                  chain identifier (A, B, C)
    resi                   residue number (or range – use a colon)
    resn                   residue name
    name                   atom name

To start over, File > Reinitialize

If you can’t find anything, show lines and then A (action) > center




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