Step 2: Edit model, XPLOR

Step 2: Edit model, XPLOR

Overview

a. Orient model properly in desired unit cell

i. Center structure at (0,0,0)

ii. Rotate coordinates to desired position

iii. Determine desired size of unit cell

iv. Shift particle center from 0,0,0 to 1/2,1/2,1/2

b. Text editing of PDB file

i. Remove header and non-coordinate lines

ii. Remove water and other non-amino acid coordinates

iii. Change "strange" atom names, such as "OXT" for terminal oxygen

iv. Renumber atoms and residues

v. Remove chain id and text from each line

vi. Add "OT" residue and "END" to end of file

c. Build molecular structure with XPLOR

i. Edit script file generate.inp

ii. Use standard XPLOR parameter files tophcsdx.pro, parhcsdx.pro, and toph19.pep

iii. Run XPLOR with generate.inp. Output files are generate.pdb and generate.psf.

iv. Check output for errors

-The program DEAL_PDB can be used to do many of the operations in steps a and b. (To download DEAL_PDB, see link on previous page.)

-Editing the coordinate file involves a trial-and-error process, and hence, may need to be repeated after the 3D map is computed in step 4.

Detailed instructions

a) Orient model properly in desired unit cell. The PDB coordinates probably need to be moved translationally, and perhaps rotationally as well. The PDB file you retrieve may not be in the convention you are used to. You should choose a convention and stick to it. The matrices you use for rotation in step ii must correspond to the convention you are using.

i. Initially, if the coordinates are not centered on (x,y,z) = (0,0,0), move them to this position. The initial center can be computed by averaging separately the x, y, and z coordinates. (To translate to [0,0,0] simply add [-average x, -average y, -average z] to the coordinates. In DEAL_PDB, use the command "translate".)

ii. Next, the coordinates need to be placed in a suitable orientation. You may desire them in an orientation different from that in the PDB file. If so, apply a rotation matrix to the PDB coordinates (In DEAL_PDB, use the command "rotate".)

iii. Finally, determine the size in Å of the unit cell you want for your low-resolution density.

iv. Add (1/2,1/2,1/2) of the unit cell to each coordinate in the file to shift the center to (1/2,1/2,1/2). (In DEAL_PDB, execute the command "translate 1/2 1/2 1/2".) Note you must supply the actual value for the center of the cell. For example, if the cell is 500X500X500, then use "translate 250 250 250" in DEAL_PDB.

b) Text editing of PDB file. The computations in step c require that some editing of the PDB file be done.

i. Remove the header and any line--except "END"--that does not contain coordinate information. (Lines with coordinate information are those that have "ATOM" at the beginning.) This may be done with a text editor. In DEAL_PDB, the header can be removed by the command "write [filename] noheader".

ii. Remove lines that have water or other non-amino acid coordinates. The simplest way to do this is with a text editor.

iii. Change "unconventional" atom names, such as "OXT" which is sometimes used for the terminal oxygen of an amino acid, to "conventional" names, such as "O" for the terminal oxygen. (It probably a better use of your time to not do an exhaustive search for such "unconventional" names. XPLOR will let you know in step c if it has a problem with a certain atom name. Then you can simply re-edit your file and rerun step c.)

iv. Renumber the atoms and residues starting with "1". (In DEAL_PDB, use the commands "renumber atom 1" and renumber residue 1".)

v. Remove the chain ID and text entries for each line. (In DEAL_PDB, use the command "set_value" followed by "chain_id" and "text".)

vi. Finally, the file should end with 1) the C-terminal oxygen, 2) an extra oxygen atom labeled "OT", at coordinates slightly different from the previous oxygen, and 3) the line "END", as in the following example for PDB format:

ATOM  20852  C   PHE  5373     252.950 402.164 482.294  1.00136.36

ATOM  20853  O   PHE  5373     252.000 401.978 483.069  1.00136.95

ATOM  20854  OT  PHE  5373     251.990 401.968 483.079  1.00136.95

END

c) Build molecular structure in XPLOR (http://xplor.csb.yale.edu/xplor-info) with generate.inp script. This is a step that must be done before the structure factors can be computed in step 3.

i. Edit generate.inp by changing "polyoma_OT.pdb" to your PDB filename.

ii. XPLOR files tophcsdx.pro, parhcsdx.pro, and toph19.pep are needed. They have standard parameters essential for adding the H atoms. They are standard files and should not be altered.

iii. From a UNIX prompt, execute the command "xplor < generate.inp > generate.out". Output files are generate.pdb, generate.psf, and generate.out (log file, if run as stated in first sentence).

iv. Check output for errors. If errors are bad enough, generate.pdb and generate.psf are not written out. Even if they are, please check the generate.out file for errors (search for "ERR"). You may need to re-edit the input coordinate file. Also, the generate.pdb file may have atoms with coordinates of 9999.0. These must not be used in the structure factor calculation. They will be deleted from the structure factor calculation by the delete option in the XPLOR script fcalc.inp (step 3).