Modeling Macromolecules

The focus of these experiments in on homology or similarity modeling. The utility of homology modeling and the general approach to it are well established. (although there is considerable controversy over the details). Other aspects of modeling such as threading or ab initio folding are still experimental and so will not be treated in these lectures.

The experiments

Accuracy of Structure

Experiment 1: Modeling dendrotoxin and BPTI

BPTI and Dendrotoxin are small proteins with moderate homology. Despite quite different biological functions (BPTI is a serine protease inhibitor, and Dendrotoxin is a neurotoxin which blocks ion channels) these proteins are quite similar in structure.

	BPTI
	Dendrotoxin

The sequence alignment between the two proteins, shown below, shows moderate homology. However, notice that all of the Cysteine residues are conserved, which suggests (correctly) that the disulfide bonds are conserved between the two proteins. Conservation of sequence patterns, like this one, can suggest homology even where it is otherwise weak. The conserved patterns are not always cysteines, but can be patterns of hydrophobic residues or active site residues.

Dendrotoxin pRrklCilhrnpGrCydkIpafyYNqKkkqCerFdwsGCggnsNrFKtiEeCrRyCig* 
BPTI        *RpdfCleppytGpCkarIiryfYNaKaglCqtFvygGCrakrNnFKsaEdCmRtCgga  

When running this experiment it is important to look at the conserved residues, the surface residues, the tyrosine and tryptophan residues which form the core structure, and the N and C-terminal residues. The strand from residues 15 to 22 is also worth examining for changes.

Required Programs

• AMMP

Required files

• bptidtx.ammp, BPTI mapped to Dendrotoxin
• dtxbpti.ammp, Dendrotoxin mapped to BPTI

Required Command files

• tsearch.ammp, Use torsion searches
• dgeom.ammp, Use Distance Geometry
• polish.ammp, Energy minimize the results
• rms.ammp, Calculate the rms error

Procedure

1. Retrieve the files
   You can't run the examples without the program or the program files.
   
   
2. Build Dendrotoxin from BPTI
   Start GRAMMP. The program will start and generate a graphic's window. This window will display the molecule and allow you to manipulate the molecule, calculate geometric properties, and run AMMP commands. AMMP commands can also be typed into the AMMP text window (the window where you typed the GRAMMP command). Read in the file bptidtx.ammp: use the ( Load AMMP file in the File menu or type read bptidtx.ammp; in the AMMP text window). You will see the atoms in common between BPTI and dendrotoxin with many bonds drawn to what appears to be an arbitrary point (which happens to be 0,0,0). These are the atoms which have to be placed to build a complete model.

   Two approaches to building these new atoms should be used for this experiment, and you should monitor the differences in the solutions. Torsion search algorithms are quite popular and are implemented in the script tsearch.ammp. An attractive alternative is to use distance geometry which is implemented in dgeom.ammp. Since it is important to understand the limits of modeling, as opposed to the limits of a particular algorithm, it is important to run both of these procedures and compare the results.

   Using tsearch.ammp tsearch.ammp builds a structure with highly ideal bond-angle covalent geometry and then searches the side chain torsion angles to find the energy minima. Read in the file tsearch.ammp and watch the GRAMMP window for the progress of the torsion search . The molecule can be moved about in the draw window with either the mouse or the scrollbars. The size of the molecule will appear to hop about because of the "autoscaling" feature. You probably want to turn this off using the clicking the Autoscale checkbox on the Controls menu. While you're at it you should play with the other controls to see what they do. You can select an atom by clicking on it. The atom name and serial number are displayed. You can calculate geometric properties using the Geometric menu. Select the Distance option and then click on two atoms. The distance (in Angstroms) will appear. Watch the distance change as the molecule is minimized. You can also select Angles (then select three atoms) or dihedral (then select four atoms). The Controls menu contains options for rotating, translating, and scaling. Note: The View menu contains options for changing the molecular representation. You can view down each axis or change the color of the molecule to Monochrome (white) or CPK (color based on atom type) or Force (color based on the force on each atom, white-small force, red-large force).
   
   

   Using dgeom.ammp tgeom.ammp performs a distance geometry calculation to build the missing atoms. This is the complement or dual of the torsion search. Instead of building a highly ideal structure and then finding the best packing, dgeom.ammp builds a well-packed structure and tries to find the closest ideal structure. Exit GRAMMP (using the Exit option on the File menu. Restart GRAMMP and read the bptidtx.ammp file again. To begin the minimization, read the dgeom.ammp file.

   Cleaning up the structure The script polish.ammp performs a short run of energy minimization with the whole potential. The use of energy minimization on a homology model is somewhat controversial, as one school of thought believes that the best model is simply to replace the side chains on residues in the conserved regions. Homology modeling provides a valuable test case for the development of potential energy force fields and therefor not performing energy minimization because some molecular mechanics force fields are poor is an unscientific choice.

   Saving the results You may want to save the results for later study. Use the Output AMMP file option on the File menu to output a new ammp file. It is a good idea to chose a new unique name for the output.

   Analyzing the results The script rms.ammp superimposes the structure on the experimental coordinates of the target. The coordinates are stored in the tether data structure of AMMP. This script uses a genetic algorithm to superimpose the structures. For highly similar structures, the genetic algorithm is somewhat wasteful, but for dissimilar structures it is an appropriate algorithm. After the superposition, you should examine the detailed differences in structure. Click on the Show Tethers checkbox on the Geometry menu. A purple dashed line is drawn between each atom and the position of the atom in the dendrotoxin target.

   Note where the largest errors are. (hint - look at the side chains on the surface and the N and C-terminals). Are they significant? Why or why not? Now color the atoms by force (View|Colors menu). Do the errors correlate with the force (which is proportionate to the residual error in the structure)? Do you see the same errors in both the distance geometry and torsion search models?

3. Build BPTI from Dendrotoxin
   The procedure for building BPTI from Dendrotoxin is identical to the procedure of building Dendrotoxin from BPTI (listed above). Start GRAMMP. Read in the file dtxbpti.ammp You will see the atoms in common between BPTI and dendrotoxin with many bonds drawn to what appears to be an arbitrary point (which happens to be 0,0,0). These are the atoms which have to be placed to build a complete model.

   Just like in the modeling building of Dendrotoxin, two approaches will be employed to build the model. The script tsearch.ammp uses a torsion search algorithm for building new atoms. While the file dgeom.ammp uses a distance geometry algorithm.

   Cleaning up the structure The script polish.ammp performs a short run of energy minimization with the whole potential.

   Saving the results You may want to save the results for later study. Use the Output AMMP file option on the File menu to output a new ammp file. It is a good idea to chose a new unique name for the output.

   Analyzing the results The script rms.ammp superimposes the structure on the experimental coordinates of the target. The coordinates are stored in the tether data structure of AMMP. After the superposition, you should examine the detailed differences in structure. Click on the Show Tethers checkbox on the Geometry menu. A purple dashed line is drawn between each atom and the position of the atom in the dendrotoxin target. Note where the largest errors are.

   Questions

   • What errors do you see?
   • Do the errors relate to the local environment of the side chain?
   • Did the two algorithms produce drastically different structures?
   • Where errors in the placement of the peptide backbone (main-chain errors) corrected?
   • Were there any obvious symptoms of error in the models? (i.e. in the absence of the experimental structure, could you have told where the errors where?)

Experiment 2: Modeling Protein G and BPTI

	BPTI
	Protein G

The sequence alignment between protein G and BPTI shows poor homology. However, there are enough similar residues (Tyr|Phe, Arg|Lys,...) that with a few short gaps a decent threading alignment could be built. In fact, the proteins do share a small sub-structure.

Protein G ***mtyklilnGktlkgettteavdaAtaekvFkqyandngvdgewtydDatkTftvte
BPTI      rpdfcleppytGpckariiryfynakAglcqtFvyggcrakrnnfksaeDcmrTcgga*  

Required Programs

• AMMP

Required files

• bptipg.ammp, BPTI mapped to Protein G
• pgbpti.ammp, Protein G mapped to BPTI

Required Command files

• tsearch.ammp, Use torsion searches
• dgeom.ammp, Use Distance Geometry
• polish.ammp, Energy minimize the results
• rms.ammp, Calculate the rms error

• AMMP
• preAMMP

Required Command files

• Use torsion searches
• Use Distance Geometry
• Energy minimize the results

Required Skills

• Use of GCG, Blast, or similar package to search for similar sequences from the pdb
  
• Access to the PDB
• Using a text editor such as vi

Procedure

1. Select a protein target Choose a protein where you don't know the structure. This can be one from your work or one that you find interesting for other reasons. While AMMP can handle quite large problems (>10000 atoms), it is probably a good idea to choose a sequence of about 100 residues (unless you can arrange to run the program overnight). Run the alignment program to find similar sequences in the Protein data bank where the structures known. Ideally you will find several structures with stretches of high sequence similarity and few insertions or deletions. The proteins should also be nearly the same size, as it is unlikely that part of a large protein will be the correct fold for a small protein unless it is a seperately folded domain.

   Use your judgement on the results. Are the alignments like the BPTI-Dendrotoxin alignment or more like the BPTI-Protein G alignment?

   If you can't find a match, or have no idea of an interesting protein run the sequence search in reverse. For example, take the sequence of Hen egg white lysozyme and find a sequence from another species. A few more exciting examples are Human growth hormone (pdb1hgu.ent), Epidermal Growth factor (pdb1egf.ent), GCSF (pdb1bgc.ent), Beta Nerve growth factor (pdb1bet.ent), CD2 (pdb1cdb.ent), CD4 (pdb3cd4.ent) and Protein G (pdb1pga.ent).
   
   

2. Change the sequence The sequence will need to be changed from the starting model to the target. Use the program newho to build a new pdb file from a parent pdb file using an alignment file. Insertions and deletions are specified with '*' in the appropriate sequence (many programs supply '.'). Newho requires a file called "table" which defines the atoms that are iso-structural for each pair of amino acids.

   The alignment file for BPTI-Dendrotoxin is:
   pRrklCilhrnpGrCydkIpafyYNqKkkqCerFdwsGCggnsNrFKtiEeCrRyCig*
   *RpdfCleppytGpCkarIiryfYNaKaglCqtFvygGCrakrNnFKsaEdCmRtCgga
   
   Edit the pdb entry file to extract the particular chain in a multi-chain entry, or the average coordinate set in an NMR structure entry. While we usually include ligands and water molecules when generating homology models, these should be removed in order to make the generation of the molecular geometry file straightforward. While multiple chains can easily be handled in AMMP, the writeup below described how to handle a single chain
   Edit the output file and replace the CYS with CSS for any cysteine in a disulfide bond. The preammp dictionary differentiates between CYS and CSS.

   Note how many residues there are in the chain because you will need this later

3. Generate the molecular geometry file preAMMP is the program which converts a Cartesian atomic description of the structure into the molecular geometry file which AMMP reads. It is necessary to match up the atoms with the appropriate terms in the potential set, and ALL molecular modeling programs do this someway or another.

   Preammp first requests a parameter file. Select the file named "atoms.sp4". It then asks to find a template. The template defines the atoms and bonds in each type of amino acid. They should be in a directory "protein", but could be somewhere else depending on how they were unziped. There will be many files in this directory, but you should see files named like "ALA", "ASP", ..., and "TRP". Open any file, because all that is being extracted is the directory name. It then asks for a pdb file. This is the file you just generated with NEWHO. Open it. Finally it asks for an output file. Supply a new file name ending in ".ammp" or ".amp".

   The program should run silently without warnings or error messages. You may receive a message about "something.OXT" not being in the geometry. This means the c-terminal residue has both atoms of the terminal acid. If that is the case you will have to add the geometry terms to link that atom into the structure (which is very easy to do).

   Linking the chain. The individual amino acid residues are not joined at this stage. It is necessary to link them. The script linkme.ammp will link all the residues. However, you must set the variable iresm. If there are N amino acids then set the value to N+1 with the command "seti iresm value;" (e.g. seti iresm 101; for a 100 residue structure). Explicitly setting the variable ires to a value, and the variable jres to a value and reading the script "peplink.ammp" (in the protein directory with the templates) will link residues ires and jres. Linkme.ammp will also attempt to link an oxt group at the cterminal.

   Disulfides must be specified by hand. Set the variable ires to one cysteine and the variable jres to the other and read "sslink.amp" (also in the protein directory). "seti ires 100; seti jres 110; read protein/sslink.amp;" will generate the disulfide between cysteines 100 and 110.

   The terminal acid group can be linked, if necessary by using the script "oxtlink.amp". (again in the protein directory with the templates). Set the variable ires to the last residue and read oxtlink.amp. "seti ires 100; read protein/oxtlink.amp;" links the c-terminal residue 100. If you used the peplink.ammp script and the variable iresm was properly set this routine will not be needed.

   All of the linking scripts check for valid atoms. You cannot create a disulfide bonds between two non-sulfur atoms, nor improperly link in a non-extant oxt atom.

   It is a good idea to save the file at this point, because generating all these linkages is a bit of a pain. You don't really want to do it again. The main window "file|output AMMP file" menu is the easiest way to do this.

4. Build the model Use the command files tsearch.ammp or dgeom.ammp which you used before. You only need to use one of them, and the choice is yours. The command files use a variable to determine the range of amino acids to build. You must set this variable. If there are N amino acids then set the value to N+1 with the command "seti iresm value;" (e.g. seti iresm 101; for a 100 residue structure).