Step 2: Edit model, CCP4

Step 2: Edit model, CCP4

Overview

a. Determine unit cell size and angles (procedure currently requires a=b=c, a =b=d =90° values)

b. Edit sfallheader file

i. Change first six columns of CRYST1 line to the a, b, c, a , b , d values from step a

ii. Set 1st column of SCALE1 line, 2nd col. of SCALE2 line, 3rd col. of SCALE3 line to 1/unit cell distance.

c. Remove non-coordinate lines with command "grep ATOM original_PDB_file > output_file"

d. Remove non-coordinate, non-amino-acid lines from the output from step c

i. Remove lines with a different format from the coordinates (remark and header lines)

ii. Remove any non-amino-acid coordinates

e. Generate symmetry-related coordinates, if needed or desired

f. Merge output from steps b and e (d replaces e if no symmetry was applied)

Detailed instructions

a. Determine unit cell parameters. View pdb file and search for a line containing SCALE1. You should see lines similar to those below:

CRYST1 305.580 360.780 299.460 90.00 92.89 90.00 P 212 2BPA 323

ORIGX1 1.000000 0.000000 0.000000 0.00000 2BPA 324

ORIGX2 0.000000 1.000000 0.000000 0.00000 2BPA 325

ORIGX3 0.000000 0.000000 1.000000 0.00000 2BPA 326

SCALE1 0.003272 0.000000 0.000165 0.00000 2BPA 327

SCALE2 0.000000 0.002772 0.000000 0.00000 2BPA 328

SCALE3 0.000000 0.000000 0.003344 0.00000 2BPA 329

These lines make up the header information you will require for sfall (step 3). The CRYST1 line describes the a, b, c, a , b , d values for the unit cell of the crystal. Currently, the programs require a triclinic unit cell that is, a=b=c, a =b =d =90° . Take a close look at the CRYST1 line for your structure, and figure out the smallest cubic cell (round up to the nearest 100) which would contain all the information (write the unit cell size down).

b. Edit sfallheader file, a header file for sfall.

i. Change the first six columns of CRYST1 line (a, b, c, a , b , d , respectively) to the unit cell parameter values you determined earlier.

ii. Change the first column of SCALE1 line, the second column of SCALE2 line, and the third column of SCALE3 line to a value of 1/unit cell distance. When changing the values, MAKE SURE YOU DON'T CHANGE THE FORMAT!!! In other words, keep all the spaces and number positions the same - changing them can cause your sfall job to die. The other columns should be zeros. Don't alter the ORIGX lines. c. Remove non-coordinate lines. At a UNIX prompt, execute the following command:

grep ATOM original_PDB_file > output_file (e.g. "grep ATOM pdb2bpa.ent > phix174_noheader.pdb")

In the phix174 example, this command will copy only those lines containing ATOM to phix174_noheader.pdb, eliminating lines you don't need for this conversion.

d. Remove non-coordinate, non-amino-acid lines from the output from step c, e.g. the phix174_noheader.pdb file.

i. Remove any lines which have a different format from the coordinates (remark and header lines).

ii. Remove any coordinates that do not include a three-letter amino acid designation--delete those lines. Only amino acid coordinates should remain.

e. Generate symmetry-related coordinates, if needed or desired. Currently, we only have programs available for icosahedrally symmetric particles. Use programs apply60f or new_mk60 (new_mk60 also requires that the file icos.mat be in the same directory as the new_mk60 executable and the input and output files). These two programs are for each of the two conventions for icosahedral coordinates. (To compile the program(s) in UNIX, use the command "f77 -o <desired executable name> <source code file>".) For example, for phix174 the apply60f program is the one to use, but other icosahedral virus pdb files may require the use of the new_mk60 program--for example cowpea mosaic virus. Unfortunately, as yet the only way to determine which program is appropriate is to go through the full conversion--if the 3D density map (step 4) has large holes in it, then the wrong program was used and you must start over with the other program. To save the time waiting on the sfall job, you may wish to run your structure through BOTH programs (and continue through the rest of the conversion with both versions). Make sure to label each uniquely.

f. Merge output from steps b and e (d replaces e if symmetry was not applied). Once apply60f or new_mk60 has finished--or was skipped--merge the sfallheader file and the altered coordinate file. The sfallheader file is placed before the altered coordinates in the new file. In UNIX with our phix174 example, use the command

cat sfallheader_phix174 phix174_apply60f.pdb > phix174_sfall.pdb

This adds the modified header to the altered pdb file. The resulting file, phix174_sfall.pdb, is the file you will be running through sfall in step 3, which generates the structure factors.