Step 4: Compute 3D density with SFs from CCP4

Step 4: Compute 3D density with SFs from CCP4

Overview

a. Execute mtz2various.sh script file to change SF format to ASCII

i. Edit mtz2various.sh to match your directories and desired input/output filenames

ii. Run edited mtz2various.sh

iii. Check output log file for errors and count the number of lines in the file

b. Shift particle center to (1/2,1/2,1/2) and change SF format with program translate_half

i. Compile translate_half, if necessary

ii. Run translate_half

c. Use EMSFORMAT_PIF program to convert ASCII-format SFs to PIF format

d. Use EMSF3DBT to compute 3D density from SFs

e. View the 3D density, repeat steps 2-4 if necessary

Detailed instructions

a. Execute mtz2various.sh script file. This converts output from sfall (step 3), which is in binary format, to ASCII format (specific format is specified by .sh file).

i. Edit mtz2various.sh to match your directories and change the input, output, and log files if you wish. You shouldn’t modify any of the other parameters.

ii. Run edited mtz2various.sh. It is best to run in batch mode. See your system manager or a competent colleague for help in running this on your system.

iii. Check the output log file for errors.

b. Use translate half program to move the particle center to (1/2,1/2,1/2) and to change the format of the SFs.

i. Compile translate_half, if necessary. In UNIX, use the command

"f77 -o translate_half translate_half.f"

ii. Run translate_half. Input is the output data file from step a.ii (the mtz2various.sh run). Output is a structure factor file in ASCII format.

c. Convert ASCII SFs (from step b) to PIF format with EMSFORMAT_PIF program. Input the unit cell value from step 2.a.

d. Compute 3D density with EMSF3DBT program. Input the output from step c and the maximum resolution desired for the map. Note that this value can not be a higher resolution than the structure factors calculated in step 3.a.iv. Use all the defaults the program offers (hit return) except for the dx, dy, dz line: in this line, choose the pixel size (in Å) for your map--this value should be less than 1/2 that of the maximum resolution (e.g. if your max. resolution is 20 Å, then the pixel size should be < 10 Å). Of course pixel size is not an arbitrary value, but should be carefully calibrated. Output is a 3D density map in PIF format.

e. Repeat step d as desired to produce maps of different resolutions and pixel sizes. Remember to limit the highest resolution to the structure factor resolution (step 3.a.iv.).

f. View the 3D map(s). Use ROBEM (http://bilbo.bio.purdue.edu/~workshop/help_robem/) program or another program to view. You may need to reverse the contrast on the map and/or rotate the map to compare it with 3D reconstruction maps. The program EMMAPZOOM makes maps at different sizes. If the result is not satisfactory you will probably need to repeat steps 2-4. Check the orientation and center of your structure. If your structure is an icosahedral virus and there are large holes in the map, then the particle requires the other program (apply60f.f or new_mk60.f, step 2.e) to generate the icosahedral particle.