Notes on using the ingle particle helical suite of software

Source

• http://emlab.rose2.brandeis.edu/helical
• publication:
• sample data: http://emlab.rose2.brandeis.edu/node/128

Notes on running (using the supplied TMV particle images)

CTF

• initial CTF determination: run ctffind3.perl
  • outputs ctffind_results.txt, which gives the CTF params for each micrograph
• run readmrc.pl
  • creates a spider docfile with initial CTF params for each micrograph
  • output file is defocus.spd
• ctftilt -- CTF determination with tilt axis
  • convert mrc images to spider, and shrink 2-fold: proc2d A00.mrc A00.spd spider-single
  • run ctftilt_wjr.spi -- window the files, convert back to mrc, create ctftilt.com scripts (one per micrograph)
    • spider spi/spd @ctftilt_wjr
  • run each of the ctftiltNN.com scripts -- this way, run all at same time to parallelize
  • run ctftilt_wjr2.spi -- convert the output from above files into a single spider docfile
    • spider spi/spd @ctftilt_wjr2 (needs to run readtilt.py, in current directory)
    • output file is defocus-tilt-pixs163.spd

Boxing

• Open each micrograph in boxer
• Box size 666
• Put a box at the start and end of each filament
• Save as ANN.box, where NN is file number
• run script split_box_files.pl, which will make a new file for each pair of boxes (each filament)
• for each micrograph:
  • temporarily move the micrograph and all associated .box files into a tmp subdirectory
  • in that directory, open the micrograph in boxer
  • Open the first box file, for the first filament
  • Set Box overlap to 606
  • Find the marked filament, and delete the pair of boxes
  • Click the "helix" button, and add a new pair of boxes which cover the filament
  • Should now get a set of overlapping boxes covering the entire filament
  • Save the box database by overwriting the .box file which you had opened
  • Clear the boxes
  • go on to next filament

Combine CTF and box information

• With box files and ctf files in same directory, run doc-angle-ctftilt.amy
  • outputs 051215_tmv-flood1x-cut-angle-ctftilt-doc20.spd