Helical Processing : Initial steps

display tube image and transform

• mrcdisp to display
• hft to calculate fft (must create id.box file with box dimensions)
• hftout or xdispf to display fft

determine repeat distance

• acf
  • used to correlate a small piece of tube (the "reference") with the remainder (the "test")
  • Do not overlap the small box with the big
  • make the small box width the inner part of tube (no edges) and the big box wider than tube
  • CCF values are scaled from 100 (highest ccf value found) to -100; generally choose peaks >=85 for testing

Examine repeat

• put acf values in box (ylength from center of small box to peak)
• after transform, use hftout to convert fft to ascii format (if out.cnt exists, hftout < out.cnt, otherwise manually run)
• layer lines should be horizontal and lie along one pixel
• use plotphase.com to aid centering: phases along equator should be 0 or 180.
  • plotphase.com {id}
  • gnuplot -- > plot "id.phase"

determine alignment of tube (dx and out-of-plane tilt)

• nltabl 60 2.729 (60 is extent along layer lines and 2.729 is pixel size in A)
  • makes id.tbl and id.id files
• lnl.com id (extracts data from id file)
• truncate_lnl.pl id (selects only "strong" layers lines for further consideration)
  • must edit truncate_lnl.pl to specify which layer lines to use
  • makes id.lnl file
• hlxsall.pl id [min points] [max points]
  • Default total number of points generated as input for srch is 12-20
  • generates id.src file, which is input for srch
• srch.com id
  • reports best parameters (omega and dx) in id.rdl file
  • can edit srch.com to control the starting values for omega,dx
  • if dx >= 1 pixel, adjust box file and run hlxsall.com, srch.com again

create layer line files and generate statistics

• Edit box file so that output is 1024 x 2048 rather than 512 x 1024
• hlxmk.pl id
  • reads id.tbl file, id.rdl file
  • writes id.hlx control file for hlxfl
• hlxstats.pl id
  • automatically does the processes listed below:

• hlxfl.com id
  • Extracts layer line data from fft to create Big G files for near and far side
  • Needs id.hlx control file, created above.
  • makes id.nea and id.far
• hlxresidual.com
  • edit id.halfit to change repeat distance (id.id) and replace ll positions with those in id.lnl
• nfavg.com id
  • averages near and far sides to produce id.avg
• hlx2fld twofold
  • edit twofold.cnt to include repeat distance

File Conversion Notes

• After Zeiss scanner on SGI:
  • use tld2mrc to conver to mrc format
  • Next, use sgi2pc to swap bytes to PC (x86) little-endian format
  • Next, modify with label (-2) option to convert from 1-byte integer to 2-byte integer

• After writing with programs that write in 4-bye float format (spider,eman) need to use -1 option of label to convert to integer

• fixing header:
  • sometimes header is messed up after file manipulation, and some programs will get wrong values (eg Unwin's hfts)
  • One way to fix is to read into spider, mirror it, write out as spider, convert to ccp4 with em2em, use label -1 to convert to integer

spider spi/spd
cp from raw
input.mrc
16    ; bits per pixel
2800 512 1    ; size
1024     ; skip first 1024 bytes (header in mrc)
2        ; endian format
N       ; fold negatives?
_1     ; output  -- temp since we need to mirror it
mr
_1
conv           ; output converted spider file
x                 ; mirrror along x (spider reads top to bottom and mrc is bottom to top)
en             ;quit spider

• • Using em2em, convert from spider format to CCP4 (not MRC).
  • Using label, -1 option, rewrite as integer2 format (scale 1)

Helical Processing: Unbending of tube

CTF Determination

• Use Toyoshima's pltctfx program to determine ctf
  • Need to have pltctf.def file, with description of microscope
  • calculate fft
  • sctravgft ID - convert to ascii sector data. Use default 5 sectors
  • pltctfx D - graphical interface to determine ctf
• Alternatively, use ctffind3.com to auto-determine ctf
  • With good images, gives results identical to pltctfx
  • Choose a box size of 128 or 256, choose a reasonable defocus range to search
  • Verify result by viewing montage.pow with v2 - data curve should match theoretical curve

Preparation of reference

• A reference dataset is needed for unbending steps
• Choose best tube, and reindex other tubes to same (n,l) symmetry

• • Be sure to check both orientations of tube for matching, in case tubes are directional
• After determine fitting parameters, use hlxaddhewz (hlxadd.id control file) to add tubes together
• Now this file is initial reference -reindex to each tube's symmetry with hreindex

Preparation for unbending

• make a refine subdirectory : mkdir refine
• in refine, need to rotate the tube image to be horizontal
  • Use axchgmrc to do this
  • X-dimension of vertical tube must be 512 or 1024
  • If header needs fixing (Unwin's hfts result makes no sense) use technique described above
• Several files are needed in this directory:
  • id.rdl -- the srch result file
  • id.id , id.tbl - made from nltabl
  • id.goodll - lisiting of good layer lines made from truncate_lnl.pl
  • id.box - the Toyshima format box file (1024 x 2048 output!)
  • id.rot - the default name for the horizontal tube image
  • reference.ren - the default name for the reindexed reference file
• The script setup-refine.pl will help copy several of these files from the above directory
• Next, make ctfplot.cnt file
  • Script is makectfplot.pl
  • it will ask for Toyshima's numbers for the defocus. Needs box file
• Now, make an Unwin formatted box file
  • Script is box2box.pl {id} {letter}
• Next files needed are ctf0.hlxdat and full.hlxdat, used for layer line extraction
  • Script to make these files is setup_hlxdat.pl
• Unzip local copied of several scripts: *

   unzip /cryoem/rice/bin/hlxunbend_scripts.zip 

• Optionally, copy ../id.halfit, and rename to {id}{letter}{extent}.halfit, where {extent } is first ctf0 radius

Unbending

• Should now have all necessary files
• To test, run the program

   searchbill0.com < {id}{letter}.box 

  • This does a single round of searching, and should create no errors
  • If there are problems, verify that all needed files are present, that path is correct (run Unwin, not Koji, programs, ./ should be first)
• Finally, unbend with following:

          > go.com id 5 a
  

  • where "5" is number of pieces to split tube into (currently 2-5) and "a" is repeat letter
    • This runs the entire procedure described in tubenotes

• Set ALLOWTOPICVIEW =

-- BillRice - 31 Jan 2008