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                     Bio 5476 Protocol for Lab Exercise #2
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(1)  Choose one of the following small, fast folding proteins, and download
     its PDB file from the Protein Data Bank at http://www.rcsb.org/.

          Protein                           PDB ID

          Albumin Binding Domain             1PRB
          Engrailed Homeodomain              1ENH
          Tryptophan Cage                    1L2Y
          Villin Headpiece                   1VII
          WW Domain of FBP28                 1E0L  (1E-zero-L)

     All of these proteins are mentioned in an interesting review article
     on the mechanism of protein folding from Bill Eaton's lab at the NIH.
     The article, Current Opinion in Structural Biology, 14:76-88 2004, is
     available on the ftp site for the course.

(2)  Create a keyfile for your protein that points to the OPLS AA/L force
     field parameters (ie, the file oplsaal.prm in the /params directory).
     Then run the "pdbxyz" program to convert the PDB file to a TINKER
     coordinates .xyz file.

(3)  Use the "xyzedit" program to move the center-of-mass of the protein
     molecule to the coordinate system origin at (0,0,0). This can be
     done using option 11 of the xyzedit program, and will create a new
     version of the .xyz file.

(4)  Copy the file "waterbig.xyz" from the /test directory into your
     working directory for the lab. This is a large, pre-equilibrated
     TIP3P water box. Use either a text editor, or option 6 of the
     "xyzedit" program to change the atom type numbers of the oxygen
     atoms from 1 to 164, and the hydrogens from 2 to 165. Confirm that
     164 and 165 are the TIP3P atom types in the oplsaal.prm file.

(5)  Run the "xyzedit" program on your protein mass-centered .xyz file.
     Use option 17 to embed the protein into the pre-equilibrated water
     box. This will write out a new .xyz file for the protein contained
     in the waterbox, with all overlapping water molecules removed. Be
     sure to add the size of the box to your keyfile. The waterbig system
     is a cube of size 36.342 Ang on a side.

(6)  Use the "minimize" program to energy minimize your protein-water
     system to a final rms gradient of approximately 1 kcal/mol/Ang (the
     exact value is unimportant; the minimization may require several
     hunderd iterations and many minutes of CPU time).

(7)  Add the "EWALD" and "RATTLE WATER" keywords to your keyfile prior
     to running dynamics. Now, start a series of molecular dynamics runs
     using the "dynamic" program. Use the Canonical statistical mechanical
     ensemble, ie, constant temperature or NVT. First, perform a preliminary
     equilibration run at some low temperature (perhaps 100K). Your final
     target is to perform a production MD run of a few nanoseconds at room
     temperature. If you use 2fs time steps for dynamics, then you should
     be able to run about one ns per day on an unloaded lab machine.

(8)  Make plots of the backbone and whole protein rms from the crystal
     structure as a function of simulation time. Do you think your MD run
     is equilibrated?

(9)  Discuss the stability of the secondary structural features of your
     protein. For example, if your protein contains an alpha helix or
     a hairpin of beta sheet, what fluctuations does this structure
     undergo on the time scale of your simulation?

(9)  Pick a couple of residues in your protein (a buried aromatic side
     chain and a solvent exposed surface residue might be good choices)
     and compute the time correlation function of the rotation of these
     side chains (for example, the chi2 angle of an aromatic residue).
     Correlation functions are discussed in Section 7.6 of Leach.

(10) What is the concentration of your protein in your periodic simulation
     system. Is this reasonable? What do you estimate would be the CPU
     requirements for using TINKER to run a 1 microsecond simulation of
     your protein in a water box large enough to afford a reasonable
     protein concentration.

(11) [OPTIONAL] Analyze the water structure in the first layer of water
     around your protein. Are there any very tightly bound waters (perhaps
     hydrogen bonds to one or more highly polar or charged protein groups)?
     Can you estimate the average residence time of some bound waters?

(12) [OPTIONAL] Run an implicit solvent simulation of your protein. Use the
     Generalized Born (GBSA) solvation model as in Lab 1. How does the
     ensemble of structures and RMSD from the crystal compare between the
     explicit water and implicit solvent simulations?