2D processing- negative stain
Contents
1. S orig XORIG YORIG resoll 1 axes 100 0 gamma RINNER ROUTER IRAD A B W ABANG S cell Lattice vectors eot CYCLE 2 MASKTRAN1 JOBB reference map Calculate highly averaged image from 1st unbent image giving high signal noise setenv IN corS image fft setenv OUT ref image msk setenv SPOTS S spots S image spt time masktrana exe lt lt eot 1 T T ISHAPE 1 CIRC 2 GAUSS CIRC OR 3 RECT HOLE IAMPLIMIT T or F 1 RADIUS OF HOLE IF CIRCULAR X Y HALF EDGE LENGTHS IF RECT S cell 9 9 9 9 200 1 A BX Y IH IKMN MX RMAX ITYPE eot bin rm f corS image fft FFTMSK FFTRANS1 JOBB setenv IN ref image msk setenv OUT refS image flt time fftrans exe 5 bin rm f ref i2. Less averaged version of original raw image distortions but has less 1 T T ISHAPE 1 CIRC 2 GAUSS CIRC OR 3 RECT HOLE IAMPLIMIT T or F S rad2 RADIUS OF HOLE IF CIRCULAR X Y HALF EDGE LENGTHS IF RECT cell 9 9 9 9 200 1 A BX Y IH IKMN MX RMAX ITYPE eot TWOFILE JOBB Multiply two files together FILE1 Complex Conjugate of FILE2 ICOMB 2 First image on stream 1 IN1 Second image on stream 2 IN2 Product output on stream 3 setenv IN1 image msk setenv IN2 refS image fft setenv OUT corelS image fft time twofile exe lt lt eot 2 2 0 0 S orig eot bin rm bin rm bin rm f FFTCOR FFTRANS3 setenv IN setenv OU 0 7 m time fftrans exe bin rm f QUADSERCHK JOB autoS image map setenv PROFDATA prof image dat time quadserc setenv PROFILE ICOMB 2 image msk ref image f boximage tmp corelS image cor image c corelS image ft UJ JOBI EE or TET B hk lt lt X cor image cor S size S cell F S nocells ot IPASS NRANGI l OUT IORIGIN OXA OYA OXB OY B m SIZE OF TRANSFORM Origin shifts to FFT s ISIZI c ISIZEX Lattice vectors NUMB ER UNIT CI 11 ELLS TO SEARCH
3. 676 RADIUS OF CORR SEARCH S orig POSN OF START SEARCH ORIGIN 0 0 IS ORIGIN N YES NO FOR DETAILED PRINTOUT 28 0 28 0 45 0 RADLIM IN PROFILE GRID UNITS eot bin rm f autoS image map bin rm f cor image cor bin mv CCPLOT PS QUAD2 PS CCUNBEND JOBB remember to change both titles on each run setenv CCORDATA profS image dat time ccunbendk exe lt lt eot S image cut 0 1 60 25 F 40 F ITYPE IOUT IMAXCOR ISTEP LTAPER RTAPER essential except TAPER 30 52 0 00001 thresh 45 TKX IKY EPS FACTOR TLTAXIS only FACTOR with nonbicubic S title SIMPLE UNBEND date S image PASS 2 cor image pic UNBENT PASS 2 ccunbende SIMPLE UNBEND S date eot bin mv CCPLOT PS CCUNB2 PS bin rm f corS image fft bin rm f profS image dat bin rm f S image fft setenv IN cor image pic setenv OUT cor image fft time fftrans exe MMBOX JOBB time mmboxa exe lt lt eot cor image fft filmno S title S date Y Use grid units Y Generate grid from lattice N Generate points from lattice 2 0 0 40 40 25 15 IPIXEL IOUT NUMSPOT NOH NOK NHOR NVERT S orig2 XORIG YORIG S resoll 1 S axes 100 0 S gamma RINNER ROUTER IRAD A B W ABANG S cell Lattice vectors eot The param
4. FFTPRELIM to allow the transform to be examined on graphics device label exe lt lt eot S scratch image fft 5 convert to intensities S scratch image int 0 eot The script fftir com runs a series of FORTRAN programs to prepare the image for processing The parameters that usually have to be changed are set as shell variables at the top of the script Enter the cutoff values that you determined What do the various FORTRAN programs in this UNIX script do Finish editing the script Run the script by typing go fftir When all programs have finished you can examine the log of the job fftirlog Some of the individual programs in the script can also be run interactively Try typing label and seeing what you can do with it in response to the prompts 2 5 Indexing a diffraction pattern You should now have a Fourier transform of the compressed image e g scr2 david image_name fft Display this with Ximdisp chosing the density limits to make the diffraction spots visible Select lattice refinement from the menu and try selecting half a dozen spots to calculate a lattice Save the results to image _name lat Hint the lattice should look something like this with lt 90 between A and k LB Display the lattice and write down all the spots visible above background in one half of the transform h k is equivalent to h k In data create a file called image_name spt and enter
5. all the spots thus HOOO Wo OB W etc 2 6 Unbending an image Edit the file com job1000 com pin csh x 2000 version Image processing for uncompressed 1000 X 1000 image NORMALLY ONLY NEED TO CHANGE THE FOLLOWING PARAMETERS FOR DIFFERENT IMAGES set date SL OL set title RC LH1 form B first try set cell 45 677 6 941 4 614 29 708 lattice from Ximdisp set image RcoreS31 0 3 set filmno 3 set saved per qian data set spots per qian data set scratch scrl PER gian set resoll 600 0 15 0 resolution for MMBOX of compressed image set axes 127 9 196 5 unit cell axes in Angstrom set gamma 90 0 set radl ESOM lst mask radius set rad2 P 2nd mask radius set smbox 487 512 487 512 26 x 26 box set vertl 35 0 35 0 coordinates for reference box in first cycle set vert2 650 350 set vert3 650 650 set vert4 350 650 set vertla 400 400 coordinates for second cycle set vert2a 600 400 set vert3a 600 400 set vert4a 400 600 set orig 500 500 origin for compressed image set orig2 500 500 origin for uncompressed FFT set size 1000 1000 size of compressed image set red 1 1 1 compressio
6. image avrg aph SKIP 1 END eof HEHEHE EE HEHEHE HH EH HE HE HH EE HH HH HE EH HE EE HE FE HE HE HE HE HH HEHE HE HE HEE EE HE FFTBIG to calculate a map of unit cell setenv HKLIN S scratch S image avrg mtz setenv MAPOUT scratch temp map Fftbig lt lt eot LABIN F1 AMP PHI PHASE W FOM PROJECTION 17 SCALE F1 1 0 0 0 RESOLUTION S resol TITLE title filmno date GRID S grid AXIS Y X Z XYZLIM S uc 0 0 RHOLIM 250 0 HKLMAX 20 eot FEFE RE HE a E FE EH RE HH EE EEE EH HR EE EEE FE TE REE E EE E E HERE EE HH HH EXTEND to extend the map extend MAPIN S scratch temp map MAPOUT scratch image map lt lt eof xyzlim S ucno 0 0 DOE bin rm scratch temp map PEPER EH HH HE HE a aE EE HE aE HE HE EE HE aE EE HE aE HE EE EE HE aE EE HE aE HE EE REEERE REER EE EE EE EE HE EO setenv PUBLIC FONT84 software ccp4 4 1 1 lib font84 dat NPO to create a contour plot npo MAPIN S scratch S image map PLOT S scratch image plt lt lt eof pluto TITLE S title filmno date CELL 64 0 64 0 100 0 90 0 90 0 120 0 MAP CONTRS 500 TO 500 BY 20 SECTNS 0 0 MODE BELOW 0 DASHED 1 0 15 0 GRID 0 1 COLOUR BLACK PLOT Z eof pluto HEHEHE EE HEHEHE EH HEHE HH HE HH HE HH HH HE EH HH HE HH HEHE HE HE HE HH HEHE HH HE EE EE HE PLOT to create a ps file of contour plot plt
7. several images bin csh x HEHEHE EE HE EH EH E E E E EH EH HH HH AE E AE FE E FE AE FE AE FE E FE AE FEAE E COM FILE TO CALCULATE A PROJECTION MAP WITH p3 SYMMETRY 4 FEFE FE E E E E E E E E E E E FE E E E E E E FE E FE E FE AE AE E E AE FE AE E E FE E AE AE FE AE AE AE AE E AEE E usually need to change only parameters that follow set image fimage_name set filmno 101 set ctf data set title image title set saved data set scratch scratch set date fdate set realuv a b set angle gamma set grid number of sampling points along a b c e g 60 60 60 set uc range of calculation in sample points for one unit cell e g 0 59 0 59 set ucno range to plot e g 0119 0 119 set resol 600 0 4 0 set kv 200 set cs 2 0 cd S scratch FEFE HE FE HE FE FE FE FE FE RRR FE ERE FE FE FE HE FE EE EEE FE E FE E FE E FE E FE E FE E E FE FE FE FE E FE FE E FE FE E FE FE FE FE E FE E E FE E FE FE E E FE AE FE FE AE E E E H ORIGTILTK Sets image to correct phase origin and applies beam tilt correction For multiple images can refine each image phase origin 16 against the phases of all the preceding images Need to repeat cards from FILM NUMBER to RESOLUTION LIMITS for each film Check SCL is set correctly Se SE Se OR time origtiltk exe lt lt eot 13 0 F F 0 realuv 100 angle 0 15 I
8. that can be suppressed by removing density outliers What density cutoffs would you use Type cd com nedit fftir com It should look like this bin csh x set imag set cutoffs set scratch set red cd S scratch FFTIMGRED setenv IN S scra setenv OUT S scra image name 68 98 scr username Wd taperedge cut O image name OD cutoffs determined from HISTO output working directory pixel compression factor for large images ff and initial pixel averaging and FFT taperedge is often need from discontinuities at d to eliminate transform stripes arising the image edge Cutoffs help to reduc 20 20 200 20 20 200 eot time taperedgek ex lt lt eot IAVER ISMOOTH ITAP JAVER JSMOOTH JTAP bin rm f YPLOT PLT label exe lt lt eot S image tap 99 3 S image cut S cutoffs eot other options t h ffects of bits of dust and hairs tch image pic tch image tap R 20 20 200 R F ry im ry cutoff over and underflows bin rm f S scratch S image tap label2 label exe lt lt eot image cut 4 average adjacent pixels image red S red compression factor eot FFTRANS Calculate Fourier transform setenv IN scratch image red setenv OUT scratch image fft fftrans exe bin rm S scratch S image red bin rm scratch YPLOT PS
9. CORDATA prof image dat time ccunbendk exe lt lt eot S image cut 0 1 60 25 F 40 F 30 52 0 00001 thresh 45 S title SIMPLE UNBEND S date cor image pic UNBENT PASS 1 eot bin mv CCPLOT PS CCUNB1 PS bin rm prof image dat FFTCORRED Compress corrected image label exe lt lt eot cor image pic deviations of correlation peaks from predicted lattice positions LITYPE IOUT IMAXCOR ISTEP LTAPER RTAPER LTABOUT LIKX IKY EPS FACTOR TLTAXIS S image PASS 1 4 average adjacent pixels cor image red S red compression factor eot bin rm f cor image pic setenv IN corS image red setenv OUT corS image fft fftrans exe bin rm f cor image red MMBOX JOBA time mmboxa exe lt lt eot cor image fft filmno S title date Y PASS 1 Use grid units Y Generate grid from lattice N Generate points from lattice 2 0 0 40 40 15 15 ITPIXEL IOUT NUMSPOT NOH NOK NHOR NVERT
10. FT filtered image X FFT reference box guivalent to convolution in real space setenv IN1 image msk setenv IN2 refS image fft setenv OUT corS image fft time twofile exe lt lt eot 2 ICOMB 2 2 0 0 S orig TORIGIN OXA OYA OXB OYB Origin shifts to FFT s eot bin rm f S image msk bin rm f refS image fft bin rm f boximage tmp FFTCOR JOBA Reverse FFT gives cross correlation map setenv IN cor image fft setenv OUT cor image cor time fftrans exe bin rm f corS image fft QUADSERCHK JOBA Use autocorrelation profile setenv PROFILE autoS image map setenv PROFDATA profS image dat to search for cross correlation peaks time quadserchk exe lt lt eot 0 7 IPASS NRANGE cor image cor size SIZE OF TRANSFOR ISIZEX ISIZEY S cell F Lattice vectors LREAL S nocells NUMBER UNIT CELLS TO SEARCH 6 6 RADIUS OF CORR SEARCH S orig POSN OF START SEARCH ORIGIN 0 0 IS ORIGIN N YES NO FOR DETAILED PRINTOUT 28 0 28 0 45 0 RADLIM IN PROFILE GRID UNITS eot bin mv CCPLOT PS QUAD1 PS bin rm f autoS image map bin rm f cor image cor CCUNBEND JOBA remember to change both titles on each run Correct lattice distortions in raw image based on setenv C
11. Notes on MRC IMAGE PROCESSING software 2000 The MRC Image Processing package is currently freely available to academic workers and although it is helpful if bugs are reported no support or instruction is available A list of users is kept so please do not just copy the system without informing the MRC Use of the programs is described in published papers which are referred to in Crowther Henderson and Smith J Struct Biol 1996 116 9 16 1 Overview and general philosophy The system of image processing programs developed by past and present members of the Laboratory is used in various forms worldwide for determining the structure of macromolecular assemblies The approach has been to study specimens with some form of symmetry such as crystals or helical and icosahedral particles Programs suitable for many kinds of 2 D and 3 D analysis have been written The philosophy has been to write largely stand alone FORTRAN programs for carrying out the various steps of processing At the present time November 2000 there are about 70 stand alone programs varying in length from a few lines to many thousands of lines of code The unifying principle has been to use a standard format MAPFORMAT for images and Fourier transforms and postscript format for graphics files These are also used in the crystallographic CCP4 system In the later stages of some of the procedures for example in high resolution electron crystallography amplitudes and phases ar
12. SPG NPRG NTL NBM ILST A B W ANG IPL MNRF 1001 0 30 7 61 FF IRUN LHMN LHMX IQMX IBXPHS NREFOUT NSHFTIN 100 DUMMY ct dummy aph S filmno title Film number title S ctf ctf S image aph F 45 0 0 0 1 TAXA TANGL IORIGT 43 1 42 3 0 0 0 07 01 0000 OH OK STEP WIN SGNXCH SCL ROT180 REVHK CTFREV S cs kv 0 52 1 84 cs kv tx ty S resol resolution limits 1 eot bin mv fort 3 scratch image merg aph FEE AE E E E E E E AE E E E EH EH AE E E E E AE E AE E FE E E AE E E E E E FE E FE AE FE E AE E E E AE E AE E E E E E FE AE E AE FE AE FE AE FE AE FE AE AE AE FE E AE E FE AE AE FE E AVRGAMPHS 2000 version has a bug This is an earlier version Vector sum of structure factors in scratch image merg aph fort 1 time LOCAL avrgamphs exe lt lt eot E TO0L7 0 01 0 01 8 S realuv S angle eot bin mv fort 2 scratch image avrg aph bin rm SUMMARY bin rm fort 1 FE TEHE TE E FE TE HEE HE FE E FE FE E FE TE HE FE TE FE FE E FE TE HE FE TE FE FE HE FE FE E FE TE FE FE FE FE TE E FE TE FE AE E FE TE E FE TE AE HE HE E E E E E E E E E E EEE EEE F2MTZ to convert aph file to mtz file All subsequent programs from CCP4 crystallography package setenv HKLOUT scratch image avrg mtz F2mtz lt lt eof TITLE S title filmno date CELL realuv 100 90 90 S angle SYMMETRY P3 LABOUT H K L AMP PHASE FOM CTYPOUT H HH F PW FILE scratch
13. dev i scratch image plt o scratch image ps HEHEHE EE HEE EE EH EH HE EE HH HE HH EHH HE EH HH EE HH FE HE HE HE HE HH HE HE HE HE HE HE EE HE exit FEFE ae EH HE a EE EH RE EE EE FE TE EE EH HR EE E E EEE ER RHEE HEHE EERE EH HH You should have determined that the image has some internal symmetry meaning that each Fourier term has more than one measurement These measurements can be averaged together In order to do this the correct phase origin and beam tilt must be set The program origtiltk exe does this and outputs a set of merged reflections for each symmetry independent Fourier term Edit the script to set your image to the correct phase origin OH OK Set the resolution to your highest estimate Run fourp3 com and examine the log file The program avrgamphs exe averages the measurements for each Fourier term From the table of phase residuals what is the overall phase residual 18 The remaining programs in fourp3 com are not from the MRC suite They are from the CCP4 X ray crystallography suite These programs use a KEYWORD input of parameters The job outputs a projection map image_name ps Try displaying or printing this The map has had symmetry applied can you print the same map without symmetry averaging of the data Try printing the map at different resolutions such as 35 25 or 20 A What can you infer about the structure of this protein complex 19
14. e put into a third standard format that of MTZ which allows direct access to the X ray crystallographic software in CCP4 Libraries of standard subroutines are available for reading writing and manipulating files A user program written to accept files in these three formats and producing output in the same three formats thus fits directly into the system with no need for any modifications to existing structures The libraries can be invoked at the linking step on a DEC Alpha or Silicon graphics under UNIX FORTRAN user codes will run under either system without modification MAPFORMAT involves each image or transform having an initial header block 1024 bytes long which specifies the type and size of the file the maximum minimum and mean densities and information about origins Most importantly it also contains a label field which provides a history of processing operations that have been applied to the image the convention is that each program adds a label containing a one line description of the operation and time of running Programs producing graphical output use PLOT2K routines and the resulting postscript format files can be sent to a laserprinter or viewed on a terminal using programs such as ghostscript or xview which may be installation dependent Thus apart from getting digitized data into the system and hard copy out the system of programs can be used as a coherent whole independent of the particularities of the installation 1 Readin
15. eters that most often need changing can be edited at the top of the script You should have obtained X Y values for your h k axes similar to those entered for the variable cell The script can be run in stages by activating any one of the goto lines A preceding prevents the following text from being interpreted as a command The whole script can be run as one job by removing all exit commands Try running the job in stages by removing for the stage in question and replacing in front of all other goto commands Run the job each time by typing go job1000 12 Examine the output in job 000 log The program mmbox exe at the start determines the amplitude and phase of each predicted spot What is the average intensity peak height of all the spots relative to the background Note this down How many spots have IQ 1 and IQ 2 Note this down You will notice various image files are created in the scratch directory at different stages and some of them are then later deleted Try examining the various files as you go along using Ximdisp What are the following images for image_name msk image_name fit image_name flt image_name cor image_name pic In stage4 you determine the lattice distortions by searching for peaks in the cross correlation map program quadserchk exe A graphical representation with distortion vectors magnified 20 times is shown in the two pages of QUAD1 PS w
16. from lattice IPIXEL IOUT NUMSPOT NOH NO K NHOR NVERT ER ROUTER IRAD A B W ABANG orig2 XORIG YORIG resoll 1 axes 100 0 S gamma RINN S cell Lattice vectors eot Can you estimate the resolution of the data 14 Run mmbox com to output all data to the resolution limit The data can be examined in data raw image_name aph What are the different columns in raw image_name aph 2 8 Determining the symmetry Run com allspace com in the same way that you ran all the other jobs above bin csh x ALLSPACEA Perform internal phase comparison between symmetry related spots for all plane groups cd data setenv IN image_name aph time allspacea exe lt lt eot ALL T T T 4000 SEARCH REFINE TILT NCYC Oi Ors 2025 0 ORIGH ORIGK TILTH TILTK 6 0 61 STEPSIZE PHASE SEARCH ARRAY SIZE 64 0 64 0 120 0 150 0 4 0 2 0 200 A B GAMMA RIN ROUT CS KV FO 7 ILIST ROT180 IQMAX eot What is the symmetry of the crystal By how much must the phase origin be corrected for in this symmetry group 15 2 9 Calculating a map Typically you would want many independent measurements of each reflection before calculating a map usually by processing several images The script fourp3 com has been set up to calculate a projection map from just one image However it could be adapted to combine data from
17. fully for error messages The example we are using here is a well behaved one other samples can be much more difficult to process 2 3 Getting started You will have to know some UNIX in order to run the programs If you don t know any don t panic you should quickly pick up a few basic commands Commands are shown in bold filenames in italic 2 3 1 Directory structure You will need to be very organised with your book keeping if you come to process many images It is a good idea to have a properly organised directory structure Type Is la to show all your files Various sub directories should have been set up com contains scripts for processing data contains various data files for reading and writing and keeping permanently scr username your scratch directory which contains the digitised image and various intermediate image and transform files 2 3 2 The raw image Display your image by typing Ximdisp and responding to the prompts lt return gt enters default values Try entering different parameters to see the effect Type header image_name mrc What is the size of the image in pixels Where is the image origin 2 4 Preparing the image for processing Using the online Fourier transform facility in Ximdisp find the best diffracting area Box out an image of 1000 X 1000 centred on the best area Save as image_name pic Type histo image_name pic showps HISTO PS The image has various pieces of dirt etc
18. g list Program documentation to be found in users mrc mrc image doc and prog V411 ccp4_ OSF1 doc Amos etal 1982 Henderson et al 1990 Unger and Jaeger 2 The practical class 2 1 Aims and objectives The aim of this class is to introduce you to the use of the MRC crystallographic image processing programs by working on a low resolution projection image of an untilted negatively stained crystal By the end of this session you should be able to Display an image or Fourier transform on the screen using Ximdisp Select an image area for processing Understand the information contained in the image header Perform various image manipulations using label Calculate a Fourier transform Index a Fourier transform Filter an image Correct for lattice distortions in an image Extract amplitudes and phases from an image transform Correct for the effects of the phase CTF Determine plane group symmetry and phase origin by comparing phases Estimate the resolution and quality of your data Merge and average multiple measurements to produce a final list of amplitudes and phases Calculate a projection map using some programs from the X ray crystallography programme suite CCP4 2 2 A warning The MRC programs are free but the price you pay is that they are not supported and there is no user manual NEVER assume that the data you output are correct without checking over them very carefully Always check very care
19. hich you can view on the screen or print out The programme ccunbendk exe corrects for the distortions and the results can be seen in CCUNB1 PS By looking at the results of mmbox exe at the end of stage4 how much has the signal to noise been improved Cycle2 performs a second round of unbending How do the filtered image and reference image differ from the first cycle 13 2 7 Determining the amplitudes and phases Type mmbox com gt amp mmbox log amp From mmbox log make a note of the central peak height and IQ values Try editing mmbox com to try different resolution ranges bin csh x Output amplitudes and phases from uncompressed FOLLOWING PARAMET unbent image ERS FOR DIFFERENT IMAGES NORMALLY ONLY NEED TO CHANGE THE set date 16 7 01 set title Highresolution map set cell 70 786 28 962 10 465 75 set image highres set filmno 101 set saved data set scratch scratch set resoll 600 0 4 0 set axes 64 64 set gamma 120 0 set orig2 2000 2000 cd S scratch MMBOX time mmboxa exe lt lt eot corS image fft filmno S title date Y Use grid units Y N 210 40 40 15 15 saved raw image aph woe lattice from Ximdisp filename film number resolution unit cell axes in Angstrom gamma phase origin Generate grid from lattice Generate points
20. mage msk LABEL FOR SMALL BOX IN CENTRE JOBB time label exe lt lt eot ref image flt boxS image flt smbox eot setenv IN boxS image flt setenv OUT autoS image cor time autocorrl exe lt lt eot 20 eot time label exe lt lt eot autoS image cor 1 autoS image map 210 310 210 310 eot bin rm f box image flt bin rm f autoS image cor BOXIMAGE JOBB Normally select smaller reference box to follow finer detail of image distortions but gives less signal noise 10 setenv IN refS image f lt setenv OUT refb image flt time boximage exe lt lt eot 4 0 0 S vertla S vert2a S vert3a S vert4a eot bin rm FFTRI setenv IN setenv OU ry E NOV f F T ERT V ERT EX ORIGIN FOR LAT ref image f FFTRANS2 refb image ref image f time fftrans exe bin rm f refbS image fl MASKTRAN2 MASKTRAN2 original COOR DS GIVEN IN GRID UNITS R ELATIVE TO 0 0 ORIGIN ER USE E G IN FOURIER TRANSFORM LE JOBB flt Fits image to be searched JOBB shows more local Signal noise setenv IN S image fft setenv OUT S image msk setenv SPOTS S spots S image spt time masktrana exe lt lt eot
21. n factor set nocells 80 80 80 80 number of cells to search set thresh 0 13 correlation threshold for unbending cd scratch MMBOX preliminary time mmboxa exe lt lt eot S image fft S filmno S title S date preliminary Y Use grid units Y Generate grid from lattice N Generate points from lattice 2 0 0 40 40 15 15 IPIXEL IOUT NUMSPOT NOH NOK NHOR NVERT S orig XORIG YORIG S resoll 1 axes 100 0 gamma RINNER ROUTER IRAD A B W ABANG cell Lattice vectors eot JOBA MASKTRAN JOBA Apply mask setenv IN S image fft setenv OUT image msk setenv SPOTS S spots S image spt time masktrana exe lt lt eot 1 T T ISHAPE 1 CIRC 2 GAUSS CIRC OR 3 RECT HOLE IAMPLIMIT T or F S radl1 RADIUS OF HOLE IF CIRCULAR X Y HALF EDGE LENGTHS IF RECT S cell 20 20 20 20 200 1 A BX Y IH IKMN MX RMAX ITYPE eot FFTMSK JOBA setenv IN image msk setenv OUT image flt time fftrans exe LABEL FOR SMALL BOX IN CENTRE to FT of compressed image Reverse FT to calculate filtered image JO BA Used to calcula referenc time label exe lt lt eot S image flt boxS image flt S smbo
22. x eot CALCULATE AUTOCORRELATION setenv IN boxS image flt setenv OUT autoS image cor time autocorrl exe lt lt eot 20 eot bin rm f box image flt EXTRACT CENTRAL 100 X 100 time label lt lt eot x P correlation peak profile EAK BOX FROM PROFILI 20 TIM OF SMALL BOX ES OV ERSAMPLED Pl autoS image cor 1 autoS image map 210 310 210 310 eot bin rm f autoS image cor BOXIMAGE JOBA Box out reference box for performing cross correlation search against filtered image setenv IN image flt setenv OUT ref image flt time boximage exe lt lt eot 4 NOVERT VERTEX COORDS GIVEN IN GRID UNITS RELATIVE TO 0 0 ORIGIN 0 0 ORIGIN FOR LATER USE E G IN FOURIER TRANSFORM S vertl S vert2 S vert3 S vert4 eot bin rm S image flt FFTREF JOBA Calculate FT of reference box setenv IN ref image flt setenv OUT ref image fft time fftrans exe bin rm f ref image flt TWOFILE JOBA Multiply two files together FILE Complex Conjugate of FILE2 ICOMB 2 First image on stream 1 IN1 Second image on stream 2 IN2 Product output on stream 3 OUT Performs F
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