Montage tomograms using Protomo

Collection

• In SerialEM, can collect montages at every tilt rather than single images
• To set up, choose File --> New Montage, choose number of pieces, and open the file
• Now, when tilt series starts, it will use this file as output, and do montages at every angle
• Note: Recommended to run image shift calibrations at the colelction mag before starting the tomogram
• Note 2: For a 2X2 montage at 2048x2048 per image, will end up with 3900x3900 images using default overlap.

Processing

• Transfer files to workstation and run setup_dirs as usual
• cd to raw/tmp
• make a list of pieces and setermine alignments:

      extractpieces TOMONAME.st TONONAME.pl
      cp /cryoem/IMOD/com/sloppyblend.com .
      perl -pi -e 's/g5a/TOMONAME/' sloppyblend.com
      subm sloppyblend

• check slopplyblend.log to be sure alignment errors are not too large
• aligned file is TOMONAME.bl, check this with imod
• errors in alignment can be fixed manually using midas, but it is probably better to jsut not include these images in the tomogram alignment

• protomo_setup.pl will find any .bl files in raw/tmp, and will extract images from these. Tilt information will still be from the st file so both are needed
• After running protomo_setup.pl, the .bl and .st files may be deleted to save a lot of space
• images will be in the ../clean directory. If 2X2 montages, they will be 3900x3900, and protomo can only load ~60 of these during alignment. They need to be boxed.
• Boxing to a size of 2900x2900 works for ~100 images (-70 to +70, 2 deg, cos interval). Quikie way to do this:

     mkdir mrc
     mkdir orig
     for f in *.img; do
     cutimage -fmt MRC $f mrc/$f
     fixheader -mrc2000 mrc/$f
     echo finished $f
     done
     mv *.img orig/
     cd mrc
     protomo_boxmon.pl
     rm *.img
     rename box img *.box
     for f in *.img; do
     cutimage -low $f ../$f
     done
     rm *.img
     cd ..

• protomo_boxmon.pl is in /usr/apps/script/protomo
  • assumes that you are boxing from 3900x3900 to 2900x2900
  • runs on mrc format files using label.exe
  • output files have extension .box
  • steps at beginning and are simply to swtich file formats to mrc and then back to protomo img
  • original size images are archived for later use in reconstruction

• remember that images are 2900x2900 for alignment; center is therefore 1449 1449 in param file
• can use box size 1300 1300 for protomo alignment (binning 2)
• output file size 3600x3600x120 works
• after running alignment script, need to adjust for original file sizes

• • fix the clean files to work on full size:

      cd ../clean
      rm *.img
      mv orig/*.img .
      cd ../align

• Set ALLOWTOPICVIEW =

-- BillRice - 25 Mar 2013

Montage tomograms using Protomo

Collection

• In SerialEM, can collect montages at every tilt rather than single images
• To set up, choose File --> New Montage, choose number of pieces, and open the file
• Now, when tilt series starts, it will use this file as output, and do montages at every angle
• Note: Recommended to run image shift calibrations at the colelction mag before starting the tomogram
• Note 2: For a 2X2 montage at 2048x2048 per image, will end up with 3900x3900 images using default overlap.

Processing

• Transfer files to workstation and run setup_dirs as usual
• cd to raw/tmp
• make a list of pieces and setermine alignments:

      extractpieces TOMONAME.st TONONAME.pl
      cp /cryoem/IMOD/com/sloppyblend.com .
      perl -pi -e 's/g5a/TOMONAME/' sloppyblend.com
      subm sloppyblend

• check slopplyblend.log to be sure alignment errors are not too large
• aligned file is TOMONAME.bl, check this with imod

• Set ALLOWTOPICVIEW =

-- BillRice - 25 Mar 2013

Montage tomograms using Protomo

Collection

• In SerialEM, can collect montages at every tilt rather than single images
• To set up, choose File --> New Montage, choose number of pieces, and open the file
• Now, when tilt series starts, it will use this file as output, and do montages at every angle
• Note: Recommended to run image shift calibrations at the colelction mag before starting the tomogram
• Note 2: For a 2X2 montage at 2048x2048 per image, will end up with 3900x3900 images using default overlap.

Processing

• Transfer files to workstation and run setup_dirs as usual
• cd to raw/tmp

• Set ALLOWTOPICVIEW =

-- BillRice - 25 Mar 2013

Montage tomograms using Protomo

Collection

• In SerialEM, can collect montages at every tilt rather than single images
• To set up, choose File --> New Montage, choose number of pieces, and open the file
• Now, when tilt series starts, it will use this file as output, and do montages at every angle
• Note: Recommended to run image shift calibrations at the colelction mag before starting the tomogram
• Note 2: For a 2X2 montage at 2048x2048 per image, will end up with 3900x3900 images using default overlap.

Processing

• Transfer files to workstation and run setup_dirs as usual
• cd to raw/tmp

• Set ALLOWTOPICVIEW =

-- BillRice - 25 Mar 2013