BESPA Manual
Contents
1. and output Technically there are no deference between a program and a script The Lua interpreter that execute them is a main infrastructure of this software system The language itself has an ordinary procedural syntax so that it is feasible for most scientist with casual programming knowledge This section introduces some of basic methods and necessary programming for the image processing It helps understanding some of image processing jobs in a script and writing a new script or modifying existing scripts for a specific purpose In current version processing for the volume data are very limited 3 1 Using a variable for an image data A script file usually state a command of data processing by calling pre defined functions One data set of image pixels is handled as a variable one cindy read sample cdy This function cindy read load image data file on memory and name it a variable one In fact this image data also contain many information accompanied with this data set so they are stored in a data container for the image data This data container is called a table data in Lua language Therefore it is good to consider the image is loaded into a container on memory which is referred as a variable one For example we can see the size of image print one nx one ny The image data size nx and ny is stored in this container a table as a property of image that can be specified by a period format These property values are2. loaded with config msy To change the first menu messages append text into msycmd intro_message There is also a graphical user interface and simple graphics facility for writing simple graphs or displaying image data Although hey are usually used in script files to add user interfaces using mouse and keyboard implemented graphical environment in BESPA is experimental Please note these specification will change in later releases Here is a brief introduction what kind and how they are used ina script This simple script will give a window with two buttons and small boxes They it will ask your mouse selection on the buttons After clicking mouse on a button the script end and print which button you selected a simple program for gui and graphics with lager interpreter 2008 Dec 8 moonscript bespa YoyDon messagebox lag dialog a lag button test A y 10 b lag button test B y 40 box lag dialogbox new messagebox box open YovMove 100 100 YovBox 10 10 YovMove 120 100 YovBox 10 10 ret box modal wait a mouse selection input from user box close The dialog box is a window with user interface items button checkbox keyboard entry box or slider box There are simple graphics library for rendering 2 dimensional primitives In addtion 3 dimensional graphics rendering is also supported with double buffering animation support They are not full featured graphics library but provide
3. shells Following message may appears when a history file is not found please ignore them just not to use history XXX dofile file not exist bespahistory xxx file not found bespahistory nil 2 1 late translational alignment of images Before classifying images of single particle images are aligned to have a particle in the center Also it is always nice to select good particles excluding pictures with neighbor objects At first a total sum of all image is calculated This blob sum image is made by adding images only with centering adjustment Only subset of the images 100 and four rotated images in 90 degrees step are averaged Then images are aligned to the blob sum at the best match in terms of cross correlation function These scores are sorted and requested numbers of upper images are selected bespa late moonscript path hnome uenoyt bistro ddlager makeup bespa msy moonscript bespa cdybase cdy module prototype late run select particle images good for the first classification by lateral alignment v08 10 27 XXX dofile file not exist bespahistory xxx file not found bespahistory nil input image output selected images a blob particle image optional the same selection as a previous file optional number of selection 1 gausian smooothing 2 optional 1 5 coarsen images for quick run 2 optional subset for making blob 100 max shift 4 For filen
4. 91 0 0249 0 1907 0 0838 0 1564 0 0952 0 0393 0 1033 DD D D da The class sum images are made without coarsening and gaussian filters These parameters are the same as command late in previous chapter This script crates some temporary files tmpimg cdy images filtered and coarsened XXXXsmx cdy the similarity matrix reused in a next run tmpgrp lua k means clustering result file tmpfre lua the factor coordinates of each data The similarity matrix is useful to change number of groups with the other parameter unchanged Other files are mostly checking purpose or they could be used for another custom script NOTE Number of group is less than specified in rare occasion if k means algorithm generate an empty group 2 2 east Euler angle search by triplet From some of the characteristic views of single particle images the projection euler angle to each images are estimated from mutual relationship between projection images of the same object For this angular reconstitution method the script tries to find many candidate set starting form some of good combination of triplet images First the sinogram of images are calculated and all cross sinogram correlation functions are saved to a file Then many triplet sets which satisfy mutual angular relationship are generated and sorted in terms of better values of the cross sinogram correlation Since the result always depends on quality of images and noises at this mom
5. a Backplane for Electron microscopy Single Particle Analysis USER MANUAL ver 0 9 release 352 2009 March 16 Yutaka Ueno AIST ABSTRACT BESPA is a software package for image and volume data processing in single particle analysis OVERVIEW Single particle analysis is a method for structural studies of protein and biological macromolecules From observed images of the isolated molecules the three dimensional volume can be reconstructed The method was well demonstrated by pioneering works with numbers of software systems available in both free distribution and commercial packages BESPA is yet another software tool for the single particle analysis to develop new algorithms and methods Two major methods are studied the reference free classification of images and the angular reconstitution technique Currently BESPA is not a complete program suite but provides some original programs for image processing and three dimensional reconstruction Our recent development and improvement will be released on the main web site http staf f aist go jp yutaka ueno bespa Current Linux version of BESPA is started from a shell command line by a startup script bespa which is in most case installed in usr local bin a directory for a shared program The body of BESPA is installed in its program directory A basically it runs in a terminal window taking a script file to do a job For a command prompt of the terminal BESPA runs the script file and g
6. ames please add extension cdy By default bespa create an image or volume data file with its own data format CDY For Gaussian smoothing filters use pixel unit for the radius parameter Radius 0 5 yield images mostly unchanged This smoothing proceeds before coarsening i e radius 1 5 to 2 5 is ideal for the case with coarsening 2 in pixels This radius parameter is the same as one used in Adobe Photoshop The same selection as a previous file is an option to make another class average image with different smoothing and coarsening conditions 2 1scla a spectral clustering of image eigenvector based As a reference free classification of single particle images at random orientation of molecule images are grouped into clusters by all pair wise similarity of images Since the similarity values are evaluated with translational and rotational alignment obtained groups are independent to in plane orientation of the molecule After clustering data an iterative alignment of member images will save image file contains averaged images for the group the class sum images bespa scla moonscript path nome uenoyt bistro ddlager makeup bespa msy moonscript bespa cdybase cdy module prototype scla run a reference fr classification of single particle images by spectral clustering algorithm v08 10 27 input cdy image a use Similarity matrix input optional output group average g cdy output group
7. called field of this table in Lua language There is also a useful function to access pixel value of image data directory print one pixel 1 1 This is a two dimensional array pixel where the bracket is used to access array data in Lua language For this type of data container some operations are defined two cindy zoom one 2 0 three cindy add one two four cindy affine one 45 1 1 rotate 45 degree then move 1 1 in x y cindy display four After two minus the rest of line become a comment in the program There is a program module cindy for image processing tasks Functions defined in this module can be called as cindy functionname parameters Functions to modify image data usually return a new image data a Lua table so they can be referred as a new variable If you do not need previous image data we can use the same variable as previous one to refer the new image data then previous data will be released off memory 3 2 Loading Multiple Images from a File Usually multiple images are saved ina single file because number of images usually becomes thousands Image loader function only load single images using index to the data starting from 1 one cindy read simple cdy 3 For programming efficiency the file pointer is also used to load files and read sucessive data ina file as a stream fp openfile simple cdy rb list Q for idx 1 10 do list idx cindy read fp end 3 3 Managing Lis
8. data gausian smooothing 1 5 optional coarsen images for quick run 2 optional number of groups 32 Sigma local scaling gaussian function 0 3 use mpi node 0 5 10 17 0 optional factor space dimension 80 save intermediate factor coordinate tmpfrc lua mov rot parameter file optional IMAGIC file is also supported if input image file with extension img or hdr are used The header file should exist in the same directory For output use filename with extension img so that the program first create the group average class sum images into tmpimv cdy then convert it to the IMAGIC V format sigma the local scaling Gaussian function is a technical term in spectral clustering It controls how far relationship of similarity will be suppressed The default value 0 3 usually works in most case However in case the result is not satisfactory other values between 0 2 to 0 8 should be tested check eigenvalues appeard in the console log If the eigenvalue fall off to zero too rapidly decrease to 0 25 Typical eigenvalues should be 1 0 for the first the second should be around 0 5 and the last is less 0 1 Please note this parameter was not well documented in literature of this algorithm eigen status 0 eigenval vectors 1 0000 0 1191 0 1014 0 1156 0 1014 0 1071 0 3290 0 0579 0 0407 0 0304 0 0222 0 0187 0 2798 0 0683 0 1217 0 1239 0 0548 0 1168 0 2121 0 1440 0 1916 0 1377 0 02
9. eference Manual of Lua ver 4 0 available on the web http www lua org manual 4 0 Additional programs used in BESPA are in a subdirectory named em of the BESPA program directory the place of installation For example a user guest installed source code of BESPA ina directory ueno then it builds the BESPA program directory at home guest ueno bistro ddlager makeup 3 6 Installing a built in command Several built in commands are configured by the file config msy in the BESPA directory This configuration file is evaluated everytime the bespa command runs The built in command is managed in the talbe msycmd command For example a new command myf to call a Lua funciton named myfunction will be set in folloing msycmd command myf lua myfunction There is a sample script file mylib msy to demonstrate adding this new command If an original script works fine the function may be installed as a command in this way During a development it is useful to use a lua program library for the script file to be edited extensively The lua program library is loaded as follows bespa I mylib msy myf The file mylib msy in the current directory will be loaded at first then the command line argument myf is evaluated to call the function just installed in the library Using script files will be much useful just included ina sample file uselib msy If this script works then it may be installed to em directory in BESPA and
10. ent obtained Euler angles set in a single search are not always correct and subject to further tests besna east moonscript path hnome uenoyt bistro ddlager makeup bespa msy moonscript bespa cdybase cdy module prototype east run euler angle search using triplet match seeds based on common line matching v08 10 25 input cdy image use cross Sinogram file optional output euler angle tmp tdr output 3D reconstruction tmp 3d shift to fit center of gravity gausian smooothing 1 5 optional circke mask radius 0 optional number of candidate 4 Maximum angular weight 10 angle step 4 The output is the list of candidate Euler angles for each images and a sample 3D reconstruction of the 1st candidate The list is sorted in terms of residual of common line matching The file format is just like followings At first images are centered with their center of gravity up to the provided range of shifts in x y direction The circular mask is also applied and saved as a file tmpimg cdy Then sinogram is calculated and saved ina file tmp csg The cross sinogram correlation function to all pairs of images are saved ina file tmp csx This file becomes quite large in size but can be reused in next search The main search is testing all possible triplet of images with good common line fit and sort them Up to the provided number of the candidates are saved Then next st
11. ep is assign the best fit Euler angles of all images to every candidate models The search is a iterative alignment of common line profile to minimize the total residual It was an implementation of simultaneous minimization algorithm by Penczek 1996 they introduced the angular weight This program asks for the minimum angular weight because if two images assigns the same or very close euler angles the weight becomes too small to disturb convergence of the loop The default value 10 is fine or 90 degree will impose totally even angular weight Please note the Euler angle definition is different from IMAGIC They can converted gamma beta alpha to be used in IMAGIC where the scan line of images is unchanged While IMAGIC takes the first scan line of image as Y coordinates while BESPA takes it as X coordinates File format conversion programs in BESPA keeps the scan line data intact without swapping xy axis 2 3 cr3d_ Create 3D volume not installed Once Euler angles to the characteristic views are assigned 3D reconstruction of the volume is performed by the weighted back projection method One of the candidates in calculated Euler angle sets can be selected in this script NOTE This command is not correctly installed in version v08 10 27 Please use a script file er3d msy o unix bespa cr3d msy opening script cr3d msy moonscript path home ueno bistro ddlager makeup bespa msy moonscript bespa cdybase cdy
12. h home uenoyt bistro ddlager makeup bespa msy moonscript bespa cdybase cdy module prototype using external program aux program home uenoyt bistro ddlager makeup em cdy2imv tmp12 cdy tmp12 imv file name tmp12 cdy comment making 2 files header and raw data tmpl2 hed tmp12 img using scale 1 base 0 new files created size 41x41 coord 5 00 2 00 5 00 2 00 size 67KB range 9 1 159 3 note euler 107 25 51 52 287 44 nz 1 size 41x41 coord 5 00 2 00 5 00 2 00 size 67KB range 30 7 169 7 note euler 129 64 81 19 171 86 nz 1 size 41x41 coord 5 00 2 00 5 00 2 00 size 67KB range 2 7 166 4 note euler 136 38 85 90 342 80 nz 1 size 41x41 coord 5 00 2 00 5 00 2 00 size 67KB range 0 0 175 3 note euler 71 94 63 60 50 98 0 nz 1 more images nz 1 nz 1 nz 1 nz 1 nz 1 nz 1 nz 1 nz 1 nz 1 nz 1 nz 1 nz 1 nz 1Tnzinztinzgtinzinzinzinzinzi1inzinzinzinzinzgtinzginzinzil nz 1 nz 1 adjust multi image header 100 3D volume program stop 3840 unix 5 note There might be a message of 3D Volume for converting images but never mind The same information is saved to the IMAGIC header file for a 3D volume or multiple 2D images A script is a program for BESPA written in the programming language Lua Usually a script is to describe flow of jobs and automate some tasks while a program is to describe processing details of each jobs Many program defines functions for a processing task with necessary input
13. ive a messages bespa a script file There are also useful built in commands to start pre defined script files For example bespa late This commnad late does a translational alignment of images It will ask parameters from a conosole Actually it runs a built in script late msy These scripts are installed in the BESPA program directory The image data file is usually stored in an original file format called CDY Usually a file extension cdy is used for the file To browse these image data or volume data simply give the file name to the bespa command line and a new window comes to display In addition to a X window display A version for Microsoft Windows is also available bespa image file cdy Several commands are started asking filenames and parameters from the console The console user interface is not very friendly so only backspace will work to edit the input line It has history file in the current directory named bespahistory saving previous parameters By typing the parameter input will stop is to call a shell command just like ls for directory listing should give a help message to be annotated more some implementation note On unix control Z usually works to suspend the program that will be revived by a shell command fg Control C will stop a program but once job started an external program it only stops the external program only bespa does not stop the process group like other unix
14. module prototype er3d run create 3d reconstruciton weighted back projection v08 10 25 input cdy image c3dwork4 mix10 cdy euler angle file tmp tdr output 3D reconstruction tmpl 3d model number among candidates ina file 1 optional make it a mirror optional padding in real space for FFT 16 optional only with xcc value more than threshold optional VMS version saved bespahistory 76 bespa cr3d run 1 cr3d run pad 16 modelno 1 imgfile c3dwork4 mix10 cdy tdrfile tmp tdr volout tmp1 3d VMS version saved tmpl 3d 2 home ueno bistro ddlager makeup em crtdrb proj c3dwork4 mix10 cdy m 1 pad 16 tmp tdr tmpl 3d model section 1 file c3dwork4 mix10 cdy sid 7 52 0 126 0 0 0 up 0 dx 0 dy 0 8 54 46 62 mask 15 5 filter 12 025 37 file c3dwork4 mix10 cdy sid 90 102 0 108 0 0 0 up 0 dx 0 dy 0 8 45 49 11 mask 15 5 filter 12 025 37 file c3dwork4 mix10 cdy sid 35 120 0 54 0 0 0 up 0 dx 0 dy 0 9 57 52 00 mask 15 5 filter 12 025 37 file c3dwork4 mix10 cdy sid 10 94 0 144 0 0 0 up 0 AXx 0 CYVHO0 e e a ba ete eas 74 mem allocation 53 save file tmpl 3d data range 0 5326 4 339 scale 26 0000 interpreter finished cr3d msy 24toimv export to IMAGIC 5 Image or volume data are exported to IMAGIC 5 format The command requires a file name to the argument o unix bespa toimv tmp12 cdy moonscript pat
15. oftware license REFERENCES 1 a first report Yutaka Ueno Katsutoshi Takahashi Kiyoshi Asai amp Chikara Sato BESPA Software Tools for Three dimensional Structure Reconstruction From Single Particle Images of Proteins Genome Informatics 1999 10 241 242 http staf f aist go jp yutaka ueno bespa bespa GIW99P11 pdf 2 reference free classification of images Yutaka Ueno Masaaki Kawata amp Shinji Umeyama 2005 Intrinsic Classification of Single Particle Images by Spectral Clustering Proc Biosignal Processing and Classification INSTICC Press Portugal 60 67 3 a web site BESPA a Backplane for Electron microscopy Single Particle Analysis 2008 Oct 31 Yutaka Ueno AIST Tsukuba Japan http staff aist go jp yutaka ueno bespa
16. s a subset of very ordinal one Please refer to the Programmers Guide for LAGER not available yet This summarize the internal functions in BESPA written in C language _cdy_add_lua _cdy_affine_lua _cdy_axis_lua _cdy_blt2d_lua _cdy_calcstat_lua cdy_circav_lua cdy_circlemask_lua cdy_covariance_lua cdy_cshift_lua cdy_cspan_cov_lua cdy_cspan_lua cdy_cspan_stat_lua cdy_fft2d_lua cdy_filter_lua cdy_flip_lua cdy_friedel2d_lua cdy_phase2d_lua cdy_superpose_lua calcproject3d cdy_cubic3d_lua cdy_fitsinogram_lua cdy_noise_lua cdy_polar2d_lua cdy_project3d_lua cdy_rotate3d_lua cdy_sinogram_lua ACKNOWLEDGMENT A software development of BESPA was first started in 1998 in AIST Electro Technical Laboratory and then a current prototype was developed through a collaboration works between CNRS and AIST In particular we wish to thank to Dr Patrick Schultz Dr Bruno Klahols and members of their laboratory at CNRS IGBMC for testing the code Current development of this work was supported by the Strategic International Cooperative Program Japan Science and Technology Agency JST Previous supports were from the Grant in Aid 08283101 Genome Science for Scientific Research on Priority Areas from the Ministry of Education Science Sports and Culture of Japan and from Real World Computing Project of the Ministry of International Trade and Industry BESPA is a software and program code developed in AIST The distribution comes with a free s
17. t Data One useful feature of Lua programming language is the list data It is called as a table data in Lua The simple list of numbers could also be used as an array just like previous example The table data are created when it needed at any place in the script list 1 2 5 6 7 list name test size 10 The list can be nested to describe hierarchical data For example the Euler angle data in BESPA is described ina hierarchical table 3 4 Operations to Volume Data Currently not many functions are available for volume data even though they can be loaded as the same way as the image data If it was used by the image processing functions the first slice of the volume is used and resulted new 2d images Available functions are follows cindy calcstat3d cindy display In this release the 3D reconstruction is calculated by an auxiliary program em crtrb There are experimental functions just like cdy_project3d cdy_rotate3d which is used in some scripts are described in C language functions later in this document 3 4 Sample Script There are sample script files in bistro ddlager samples to demonstrate basic image manipulations 3 5 Calling an External Program There is a function to launch an external program in BESPA which is actually in the standard programming library in Lua using the function provided by the operating system execute For other programming library implemented in Lua please refer to the R
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