Resources for the lecture material:
 Texts: Garrett and Grisham, Biochemistry, 2nd ed , 1999
               Brandon and Tooze Introduction to Protein Structure, 2nd ed, 1999
 Supplementary text material:
        (available as Acrobat files at the class Web Site):
   (Download Acrobat Reader)
        Garrett and Grisham, Biochemistry, 2nd ed , 1999
       Voet and Voet, Biochemistry, 2nd ed., 1995
        Lehninger, Nelson, & Cox, Principles of Biochemistry, 2nd ed., 1993
        Alberts et al Molecular Biology of the Cell, 3rd edition, 1994
        Zubay, Biochemistry, 3rd ed, 1993
        Garrett and Grisham, Biochemistry, 1st ed, 1993
        Brandon and Tooze, Introduction to Protein Structure, 1st ed, 1991
        Watson et al, Recombinant DNA, 2nd ed, 1992
 Research Papers: Available as Acrobat files on the class web site
 Protein structures:
   Web resources in Chime/Morpher
        PDB files in Rasmol/Chime/Kinemage
        SCOP database files in Rasmol/Chime
Other resources will be made available as needed.
 
 

WEB RESOURCES

Required for class:
   Computer connected to the internet (the faster the connection the better)

        Rasmol 2.7.1 download and manual HERE
          General Rasmol/Chime homepage HERE
               Hypertext reference manual for version 2.6.2  (HERE)

Pymol main page is HERE
         Downloads for Pymol can be found HERE (see instructor lecture
                  intro page for educational product username and password)
         A Pymol Wiki can be found HERE
         The users manual and reference manual for Pymol is HERE
         An older tutorial for Pymol HERE
         A reference card for Pymol HERE

King and Kinemage download and manual HERE

BOOKMARK THESE SITES

Protein Structure Database (PDB) (at RCSB HERE)
Structural Classification of Proteins Database (SCOP) HERE

You will need to learn how to use these resources

Example download:

Search for Phosphofructokinase R-state structure in Search-Lite or SearchFields ("phosphofructokinase and r-state").

Find the entry ID code corresponding to the R-state structure: 1pfk

Call up the entry code: it pulls up the entry summary

Lots of information in the entry summary:

What is the structure?

View the structure in King, Jmol, or Webmol viewer:
     Select View Structure on the left
     Select any of the three viewers: after loading the applet it will display the structure in the viewer window
     (each viewer gives a slightly different view)
     Finally, try opening in RasMol (a stand-alone program that does not act as a Browser plug-in)
                    Download the PDB file:
                            1) Select RasMol Viewer in the Display Molecule menu and the PDB file will be
                                 opened directly in RasMol (if it is configured correctly)
                            2) Select the Download Files on the left as "PDB text"
                                           Open downladed file using the standalone RasMol program

Either way the file will be downloaded to the computer either directly to RasMol or to disk. If there are any problems in opening the file directly in Rasmol, then save the file to disk and open it from within RasMol.

When you open the PDB file the structure should appear on the Rasmol screen

Before anything else: at the menubar -> File -> Save as -> save the PDB file to disk.
 

What are the ligands associated with the PFK structure and which residues do they interact with?

    At the menubar ->Display: cartoons

                                Colours: structure (Forms a colored ribbon structure)

Try rotating the molecule using the mouse and the mouse button.
    (Note by the symmetry that there are two subunits)

Type in the command line window:

       restrict 1-1000:a (return) (subunit B disappears)

       center selected (return) (rotation will be around the A subunit)

       select hetero and not water (return)
       Menubar -> Display: spacefill Colours: CPK
               ("hetero" atoms appear, including ADP and F-1,6-bP)
 
 

What does F-1,6-bP interact with?

You can select the atoms that are within a certain distance
        from another molecule.

Type the following:
     restrict 1-1000:a, 323:a, 324:a, 326:a
     zoom 300

Use the mouse to center the F-1,6-bP molecule

Type the following:
        select within(4.0, 323:a) and not 323:a and not water
     wireframe 0.2
     color green

Use the mouse to select each green highlighted wireframe and get the
    residue number: write it down.

Type :
          select 72:a, 125:a, 127:a, 170:a, 169:, 222:a, 249:a, 252:a, 11:a, 171:a, 103:a
     wireframe 0.3

These are the amino acids within 3.5 angstroms of the F-1,6-bP molecule.

Use the up arrow to scroll up to the typed statement or type:

     select within(4.0, 323:a) and not 323:a and not water (press return)

Type:
     dots 1000

Now you are displaying the vander Waals radii of the interacting atoms
 
 

Conclusions about PFK

Composed of two parallel a/b open sheets

Reaction catalyzed: ATP + F-6-P <-> ADP + F-1,6-bP

The products are present in the crystal structure indicating the position of the active site

Note: binding site for ADP (product) is at the COOH end of one parallel beta sheet.

Binding site for F-1,6-bP is at the COOH end of the other antiparallel beta sheet

Also note: the binding site for the allosteric activator ADP is at the
    bottom end of the domain that binds the F-1,6-bP

Later in the lectures we will compare this structure with the T-state form of
    PFK to look at how the enzyme is regulated.

IT WOULD BE GOOD PRACTICE TO DO IT YOURSELF FIRST
 

Now try a different program: Pymol

Download the program from the link above.

Open 1PFK in Pymol: it appears in wireframe

Most of the manipulation occurs through the pull-downs from the colored boxes on the right of the view panel:
A: actions
S: show
H: hide
L: label
C: color

Select Actions -> Presets -> simple (no solvent) : C-alpha display + hetero as stick form

Select Actions -> Presets -> b-factor putty: displays by color and tube size based on b-factor (flexibility)

Select Actions -> Presets -> Ligands (h-bonding of hetero atoms to protein backbone)

Select Actions -> Presets -> Ligand sites -> solid (surface representation of ligand binding sites)
check transparent, dots, and mesh.

Select Actions -> Presets -> Pretty (ribbon diagram with ligands in stick form)

Select Actions -> Presets -> Publication (Molscript ribbon diagram with ligands in stick form)

Try: main window pull-downs ( Display -> Background -> White)

Wizard -> demo (try all of them to see what Pymol can do)

You can also do command line selections:
select stuff, (hetatm and not resn HOH)
show sticks, stuff
color red, stuff
select chainA, (A//)
hide lines, chainA
hide nonbonded, chainA
show cartoon, chainA

Pymol can also create complicated movies to display protein function: Example HERE

Try making a movie:

Execute the following commands one at a time in Pymol:
viewport 640,480
mclear
bg_color white
set depth_cue=0
mset 1,100
movie.rock 1,100,90
mplay

Now save the movie using the File -> Save Movie As -> Quicktime (use Compression -> Photo - Jpeg, this can be imported into Powerpoint as a movie)

Try importing into a Powerpoint file (Insert -> Movies and Sounds -> Movie from file)
 

Practice on other structures

It is strongly recommended that you practice downloading
    structures and use Rasmol to find interesting features of the proteins.

Suggested structures to examine:

1jst (ser/thr kinase) VS. 1fgk (tyr kinase)

1btx (trypsin) VS. 4est (elastase)

1bmf (F1 Atpase)

Remember: the the use of Rasmol will be required for the
homework assignments. Get used to downloading and using
the program NOW.