2D crystallography lab
Contents
1. ICOMB 2 2 0 0 S orig IORIGIN OXA OYA OXB OYB Origin shifts to FFT s eot bin rm f image msk bin rm f ref 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 cor image fft exit stage4 QUADSERCHK JOBA Use autocorrelation profile to search for cross correlation peaks setenv PROFILE autoS image map setenv PROFDATA profS image dat time quadserchk exe lt lt eot 0 7 IPASS NRANGE cor image cor S size SIZE OF TRANSFORM 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 t Correct lattice distortions setenv CCORDATA prof image dat time ccunbendk exe lt lt eot image cut 0 1 60 25 F 40 F ITYPE 30 52 0 00001 0 13 45 S title SIMPLE UNBEND corS image pic UNBENT PASS 1 eot bin mv CCPLOT PS CCUNB1 PS bin rm profS image dat S date FFTCORRED Compress corrected image label exe lt lt eot cor image pic IO2. ISHAPE 1 CIRC 2 GAUSS CIRC OR 3 RECT HOLE IAMPLIMIT T or F ili 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 ITYPI eot bin rm f cor image fft FFTMSK FFTRANS1 JOBB setenv IN ref image msk setenv OUT ref image flt time fftrans exe bin rm f ref image msk LABEL FOR SMALL BOX IN CENTRE JOBB time label exe lt lt eot refS image flt lk box image flt S 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 auto image cor i auto image map 21073107210310 eot bin rm f box image flt bin rm f auto image cor BOXIMAGE JOBB Gl Normally select smaller reference box to follow finer detail of image distortions but gives less signal noise setenv IN refS image flt setenv OUT refbS 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 vertla S vert2a S vert3a S vert4a eot bin rm f refS image flt FFTREF FFTRANS2 JOBB setenv IN refbS image flt setenv OUT refS image fft time fftrans exe bin rm f refbS image flt MASKTRAN2 MASKTRAN2 original image to be searched JOBB 10 Le
3. scratch image avrg aph SKIPI END eof HEHEHE EEE HEE HE HE EEE HE HE HH HE HH EH HE HH HH HE HE FE HE EH HEE EE HH HEE HE EE HH HE HE HH HE FFTBIG to calculate a map of unit cell setenv HKLIN scratch S image avrg mtz setenv MAPOUT S scratch temp map Fftbig lt lt eot LABIN F1 AMP PHI PHASE W FOM PROJECTION SCALE F1 1 0 0 0 RESOLUTION resol TITLE S title filmno date GRID S grid AXIS Y X Z XYZLIM S uc 0 0 RHOLIM 250 0 HKLMAX 20 eot 17 ata a a a a a a a a a a a a a a a a a a EE aa aaa aaa aaa EXTEND to extend the map Se SH OSE extend MAPIN S scratch temp map MAPOUT scratch image map lt lt eof xyzlim S ucno 0 0 eor bin rm scratch temp map Heat E TE ae aE E TE FE AE E TE TE aE FE E TE FE FE E TE TE FE HE E aE EE FE E HE AE AE AE TE HE AE aE E E E E E E E E E E a aE EE EE E E EER Ea aE EE EE HE H 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 S 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 HH HEE HE HH EH HE HH HH HE HH HH HE EH HH HE HH FE HE HH HE EE HH HEE HE HE HHH EE
4. set rad2 set smbox set vertl first cycle set vert2 set vert3 set vert4 set vertla set vert2a set vert3a set vert4a set orig set orig2 set size set red set nocells cd S scratch goto stage2 goto stage3 goto stage4 goto cycle2 80 80 80 80 number of cells to search MMBOX preliminary time mmboxa exe lt lt eot S image fft filmno S title S date preliminary Y Use grid units Y Generate grid from lattice N Generate points from lattice 200 40 40 15 15 TPIXEL IOUT NUMSPOT NOH NOK NHOR NVERT l orig XORIG YORIG resoll 1 axes 100 0 gamma RINNER ROUTER IRAD A B W ABANG S cell Lattice vectors eot JOBA MASKTRAN JOBA Apply mask to FT of compressed image setenv IN image fft setenv OUT image msk setenv SPOTS 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 Reverse FT to calculate filtered imag setenv IN image msk setenv OUT image flt time fftrans exe exit stage2 LABEL FOR SMALL BOX IN CENTRE JOBA Used to calculate reference correlation peak profile time label exe lt lt eot S image flt box im
5. HE RHE PLOT to create a ps file of contour plot pltdev i scratch image plt o scratch image ps HEHEHE EE HHH HE EH HEHE HE HH HH HE HH EH HE EH HE HE HH FE HE HE HE HE HH EE HE HE HE EE EE EE exit HEHEHE EE HE E AE EH EH EE E E E E EH HH E E E AE E E E E HH E E HH AE E E AE FE E FE E E AE FE AE FE E FE E AE E E AE FE E FE E AE AE E E E AE FEFE F 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 and beam tilt tx ty 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 do you estimate the true resolution to be Tf this is not as high as your earlier estimate from mmbox og can you think of any reasons why 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 scratch highres ps Try displaying or printing this 18 The map has had
6. 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 prof image dat time ccunbendk exe lt lt eot image cut 0 1 60 25 F 40 F ITYPE IOUT IMAXCOR ISTEP LTAPER RTAPER essential except TAPER 30 52 0 00001 0 13 45 IKX IKY EPS FACTOR TLTAXIS only FACTOR with nonbicubic title SIMPLE UNBEND date image PASS 2 corS image pic UNBENT PASS 2 ccunbende SIMPLE UNBEND S date eot bin mv CCPLOT PS CCUNB2 PS bin rm f cor image fft bin rm f prof image dat bin rm f image fft setenv IN cor image pic 11 setenv OUT corS image fft time fftrans exe MMBOX JOBB time mmboxa exe lt lt eot cor image fft filmno S title date Y Use grid units Y Generate grid from lattice N Generate points from lattice 200 40 40 15 15 ITPIXEL IOUT NUMSPOT NOH NOK NHOR NVERT S orig2 XORIG YORIG resoll 1 axes 100 0 gamma RINNER ROUTER IRAD A B W ABANG S cell Lattice vectors eot The parameters 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
7. 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 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 HIGH RESOLUTION projection image of an untilted UNSTAINED crystal You cannot expect to be an expert at the end of this session so you should spend some time with an experienced user if you intend to use the programs in your research Unfortunately there is unlikely to be any time for us to go into calculation of a 3D structure By the end of this session you should be able to Display an image or Fourier transform on the screen using Ximdisp 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 th
8. symmetry applied can you print the same map without symmetry averaging of the data Try printing the map at different resolutions such as 20 7 or 4 A Try applying an artificial temperature factor in fftbig by editing SCALE F1 1 0 temp factor e g 150 What is the effect What can you infer about the structure of this protein complex 19
9. 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 If you do require instruction from someone the file image2000 doc outside_users doc contains a list of users so you should find someone close to you and ask to visit them so that you can learn from them Please contact Jude Smith jms mrc lmb cam ac uk 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 fo
10. S cs kv filmno eot aph aph HEHEHE EE EEE E E E E E E EH EH EH HH E E E AE E E AE AE AE AE FE AE AE AE E E FEAE E CTFAPPLY HEHEHE HE HH E E EH EH EH EH FE E AE E FE AE E E AE E E E AE AE FE AE AE AE AE E E E FEAE E CTFAPPLY Takes the original image and applies a CTF correction using defocus and input amplitudes and phases AX AY BX BY ISIZE DSTEP XMAG image DF defocus DFMID1 DFMID2 ANGAST CS KV S date title 14 This corrects for the effects of the ctf The results can be displayed or printed from scratch CTFPLOTPS Does the defocus and astigmatism seem to be correct If not try different values The corrected data are written to data ctfhighres 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 ctfhighres aph time allspacea exe lt lt eot ALL T T T 4000 SEARCH REFINE TILT NCYC One OG Ore 20 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 and beam tilt be corrected for in this symmet
11. T NOH NOK NHOR NVERT S saved rawS image aph S orig2 XORIG YORIG S resoll 1 S axes 100 0 gamma RINNER ROUTER IRAD A B W ABANG S cell Lattice vectors eot 13 Can you estimate the resolution of the data Run mmbox com to output all data to the resolution limit The data can be examined in data rawhighres aph What are the different columns in rawhighres aph Examine the file com ctfapply com image size magnification pixel size in microns spherical aberration voltage defocus and astigmatism angle axes from Ximdisp gamma bin csh x Hatt aH a aE HH AE E HE aE aE EE EE EE HE EE E E E E E EE CORRECT RAW AMPLITUDES AND PHASES FOR CTF Hat E TE He aE HH HE aE aE EE TE aE HE EE HE EE E E EE EE need to change only parameters that follow set image highres set filmno 101 set title high resolution image set data data set date 1607 01 set size 4000 set magni 62060 set step Ny OM set cs 2 0 set kv 200 0 set defocus 3900 4600 40 0 set cell 70 786 28 962 10 465 75 572 set realuv 64 0 64 0 a b set angle 120 0 set scratch scratch cd S scratch HERR AE REET EERE EERE E AE EEE EE EEE HERE TREE EEE EEE EEE EEE EE EEE setenv IN S data rawS image setenv OUT S data ctf image time ctfapplyk exe lt lt eot S cell S size S step magni S defocus
12. UT IMAXCOR ISTEP LTAP IKX IKY EPS FACTOR TLTAXIS o change both titles on each run in raw image based on deviations of correlation peaks from predicted lattice positions ER RTAPER LTABOUT image PASS 1 4 average adjacent pixels cor image red S red compression factor eot bin rm f cor image pic setenv IN cor image red setenv OUT cor image fft fftrans exe bin rm f cor image red MMBOX JOBA time mmboxa exe lt lt eot cor image fft filmno Y Y N 200 40 40 15 15 S orig resoll 1 axes 100 0 gamma cell eot exit cycle2 HH HH HH HE HH HE aE EE EE HE HE EE HH HH HH HH EH EH HE EE EE EE HH EE HE HE EE S title date f f f f f PASS 1 Use grid units Generate grid from lattice Generate points from lattice IPIXEL IOUT NUMSPOT NOH NOK NHOR NVERT XORIG YORIG RINNER ROUTER IRAD A B W ABANG Lattice vectors at a a a a a a a a a E a a a RR ERR ERR tat a a et et a a a a a a a a a a aE a CYCLE 2 HERE EEE EE EEE EEE EE EEE E E EEE EEE EEE EEE EEE EEE EEE EEE EEE HERE E E E EERE EEE EE EEE EE EERE EERE EE EERE EERE EEE EEE EEE EEE EEE EE EEE MASKTRANI1 JOBB reference map Calculate highly averaged image from lst unbent image giving high signal noise setenv IN corS image fft setenv OUT ref image msk setenv SPOTS S spots image spt time masktrana exe lt lt eot L T T
13. age flt S smbox eot CALCULATE AUTOCORRELATION PEAK OF SMALL BOX 20 TIMES OVERSAMPLED setenv IN boxS image flt setenv OUT auto image cor time autocorrl exe lt lt eot 20 eot bin rm f box image flt EXTRACT CENTRAL 100 X 100 BOX FROM PROFIL time label exe lt lt eot auto image cor 1 autoS image map 210 310 210 310 eot bin rm f auto image cor Fl BOXIMAGE JOBA Box out reference box for performing cross correlation search against filtered image setenv IN S image flt setenv OUT refS 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 exit stage3 FFTREF JOBA Calculate FT of reference box setenv IN refS image flt setenv OUT refS image fft time fftrans exe bin rm f refS image flt TWOFITLE 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 FFT filtered image X FFT reference box equivalent 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
14. ay the image by typing Ximdisp and responding to the prompts lt return gt enters default values Try entering different parameters to see the effect The image should appear featureless to the eye this is typical of cryoEM images Type header highres pic What is the size of the image in pixels Where is the image origin 2 4 Preparing the image for processing Type histo highres pic showps HISTO PS The image has various pieces of dirt and scratches 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 image highres image name set cutoffs 68 98 OD cutoffs determined from HISTO output set scratch scratch working directory set red 2 2 1 pixel compression factor for large images cd S scratch from discontinuities at the image edge setenv IN scratch image pic setenv OUT scratch image tap time taperedgek ex lt lt eot 20 20 200 TAVER ISMOOTH ITAPER 20 20 200 20 20 200 JAVER JSMOOTH JTAPER eot bin rm f YPLOT PLT label exe lt lt eot S image tap 99 other options 3 cutoff over and underflows S image cut S cutoffs eot bin rm f S scratch S image tap label2 label exe lt lt eot S image cut 4 average adjacent pixels S image red S red compression factor eot FFTRANS Cal
15. culate Fourier transform setenv IN scratch image red setenv OUT scratch image fft FFTIMGRED taperedge cutoff and initial pixel averaging and FFT taperedge is often needed to eliminate transform stripes arising Cutoffs help to reduce th ffects of bits of dust and hairs fftrans exe bin rm S scratch S image red bin rm scratch YPLOT PS 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 fftir com gt amp fftir log amp When all programs have finished you can examine the log of the job fftir log 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 scratch highres fft Display this with Ximdisp chosing the density limits to make the diffraction spots visible Select lattice refinement from the menu and
16. e 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 carefully 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 have been set up com contains scripts for processing data contains various data files for reading and writing and keeping permanently scratch contains a digitised image and various intermediate image and transform files Some documentation on the programs can be found in software image2000 doc and software ccp4 4 1 1 doc 2 3 2 The raw image Go to the scratch directory by typing ed scratch The image is called highres pic This is a low dose image of a fragment of crystalline spore coat of a bacterium embedded in glucose and preserved at 160 C Displ
17. le 0 15 ISPG NPRG NTL NBM ILST IRUN LHMN LHMX IQMX IBXPHS NR 16 against the phases of all the preceding images Need to repeat cards ESOLUTION LIMITS for each film Check SCL is A B W ANG IPL MNRF EFOUT NSHFTIN S filmno title Film number title S ctf ctf S image aph E 4Sa 000I 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 E E E E E E EEE E E E E E E E E AE E E AE E E E E E E E E FE E E E E E E E E E E E E E FE E E E E E FE E FE E E E FE E FE E E AE FE AE AE AE AE AE E E FE E FEAE 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 F 1001 0 01 0 01 8 realuv angle eot bin mv fort 2 scratch image avrg aph bin rm SUMMARY bin rm fort 1 Hae FE E FE EE HE FE HE HEE HE HEE HEE HEE HE FE FE E EH EE FE TE HE EEE E FE TE AE HE HE HE E E E E E E E E EEE HEH F2MTZ to convert aph file to mtz file All subsequent programs from CCP4 crystallography package setenv HKLOUT scratch S image avrg mtz F2mtz lt lt eof TITLE S title filmno S date CELL realuv 100 90 90 S angle SYMMETRY P3 LABOUT H K L AMP PHASE FOM CTYPOUT HHH F PW FILE
18. n CCUNBI1 PS 12 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 Why is the final Fourier transform scratch corhighres fft larger than the one you started with scratch highres fft 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 unbent image NORMALLY ONLY NEED TO CHANGE THE FOLLOWING PARAMETERS FOR DIFFERENT IMAGES set date 16 7 01 set title Highresolution map set cell 70 786 28 962 10 465 75 572 lattice from Ximdisp set image highres filename set filmno 101 film number set saved data set scratch scratch set resoll 600 0 4 0 resolution set axes 64 64 unit cell axes in Angstrom set gamma 120 0 gamma set orig2 2000 2000 phase origin cd S scratch MMBOX time mmboxa exe lt lt eot corS image fft filmno S title date Y Use grid units Y Generate grid from lattice N Generate points from lattice 210 40 40 15 15 IPIXEL IOUT NUMSPO
19. r 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 are 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
20. ry group 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 com fourp3 com has been set up to calculate a projection map from just one image However it could be adapted to combine data from several images 15 bin csh x REE EE a EE EE EH HH HE EH HHH EE HERE EE HHH EEE HH HH COM FILE TO CALCULATE A PROJECTION MAP WITH p3 SYMMETRY Hae REE HE a EEE EE HE EH HH EH HERE EH HH EEE HH HH usually need to change only parameters that follow set image highres set filmno 101 set ctf data set title high resolution image set saved data set scratch scratch set date 17 7 01 set realuv 64 0 64 0 set angle 120 0 set grid 60 60 60 set uc 0 59 0 59 set ucno 0 119 0 119 set resol 600 0 4 0 set kv 200 set cs 2 0 cd S scratch ORIGTILTK beam tilt correction For multiple images from FILM NUMBER to R Sets image to correct phase origin and applies can refine each image phase origin HEHEHE EE EE EE EE E E EH EEE EH EH E E EE E AE FE E FE E FE E E E AE E AE E FE E FE AE FE AE FE AE FE AE AE E FE AE FE AE FE AE AE AE FE AE AE E FE AE AE AE E set correctly time origtiltk exe lt lt eot 13 0 F F 0 realuv 100 1001 0 30 7 61 FF 100 DUMMY ctf dummy aph ang
21. ss averaged version of original raw image shows more local distortions but has less signal noise JE 4E SE HE HE setenv IN S image fft setenv OUT image msk setenv SPOTS S spots image spt time masktrana exe lt lt eot 1 TT ISHAPE 1 CIRC 2 GAUSS CIRC OR 3 RECT HOLE IAMPLIMIT T or F rad2 RADIUS OF HOLE IF CIRCULAR X Y HALF EDGE LENGTHS IF RECT S cell 9 9 9 9 200 1 A BX Y 1IH 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 OUT JE Se Se HE setenv IN1 image msk setenv IN2 refS image fft setenv OUT corelS image fft time twofile exe lt lt eot 2 ICOMB 2 2 0 0 S orig IORIGIN OXA OYA OXB OYB Origin shifts to FFT s eot bin rm f image msk bin rm f ref image fft bin rm f boximage tmp FFTCOR FFTRANS3 JOBB setenv IN corel image fft setenv OUT cor image cor time fftrans exe bin rm f corel image fft QUADSERCHK JOBB setenv PROFILE auto image map setenv PROFDATA prof image dat time quadserchk exe lt lt eot 0 7 ITPASS NRANGE cor image cor S size SIZE OF TRANSFORM ISIZEX ISIZEY S cell F Lattice vectors S nocells NUMBER UNIT CELLS TO SEARCH 6 6 RADIUS OF CORR SEARCH
22. 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 job4000 com gt amp job4000 log amp Examine the output in job4000 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 scratch 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 highres msk highres flt refhighres fit corhighres cor corhighres 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 QUAD PS which you can view on the screen or print out The programme ccunbendk exe corrects for the distortions and the results can be seen i
23. try selecting half a dozen spots to calculate a lattice Save the results to highres lat Hint the lattice should look something like this with about 60 between A anf k DE 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 highres spt and enter all the spots thus FOO Ore Wa OB W etc 2 6 Unbending an image Edit the file com job4000 com bin csh x Image processing for compressed 4000 X 4000 image NORMALLY ONLY NEED TO CHANGE THE T U7 T01 Highresolution map FOLLOWING PARAMETERS FOR DIFFERENT IMAGES 70 786 28 962 10 465 75 572 lattice from Ximdisp highres TO 2 data data scratch 600 0 6 0 resolution for MMBOX of compressed image 64 64 unit cell axes in Angstrom 812020 hob lst mask radius ho 2nd mask radius 987 1012 987 1012 26 x 26 box 850 850 coordinates for reference box in w50 85 0 1150 1150 850 1150 900 900 coordinates for second cycle 1100 900 1100 1100 900 1100 1000 1000 origin for compressed image 2000 2000 origin for uncompressed FFT 2000 2000 size of compressed image WD Dae compression factor set date set title set cell set image set filmno set saved set spots set scratch set resoll set axes set gamma set radl
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