ESCET Version β0.3 User Manual
Contents
1. A aset_read tset 3 ifile 1AMA pdb sele name CA aset_read tset 4 ifile 1TAS pdb sele name CA and chainid A aset_read tset 5 ifile 1TAT pdb sele name CA and chainid A Generate standard uncertainties for all models For none in the pdb file So we have put in various numbers l of the models all the necessary information can be found manually from the files or from the paper 7AAT aset_egen sset 1 esd_model dpiu dmin 1 9 npar 27968 1TAR aset_egen sset 2 esd_model dpiu nobs 36893 npar 26320 cp1 88 5 dmin 2 2 AMA aset_egen sset 3 esd_model dpiu dmin 2 3 npar 13892 nobs 17538 cp1 94 4 9 1TAS aset_egen sset 4 esd_model dpiu npar 25408 nobs 17636 cp1 87 9 rfree 30 0 dmin 2 8 ATAT aset_egen sset 5 esd_model dpiu npar 25408 nobs 18194 cp1 97 0 dmin 3 0 rfree 30 0 ddm setl 1 5 esd_scaled on lolim 2 0 hilim 5 0 ssplot on rbplot on J stop The first run on all the models is used to decide which conformers are identical For this purpose a table is printed in the log file Pairwise Comparison of entire models The following table contains for every pair of models the percentage of elements in the error scaled difference distance matrix that are smaller than the threshold value of lolim 2 00 If for a pair of models this value is larger than 98 0 the two models can be considered to be identical For an explanation2. e Two keywords patchlen and minfraglen are now available to adjust the polishing of a solution of the rigid body search See escet_ref html for details e Diagnostics for automatic consistency checks has been improved 23 Version 6 0 2d 11 Apr 2002 e Some problems with automatic consistency checking of atom lists were fixed e The Pairwise comparison table now contains a line with the mean estimated error for the atoms that are actually compared This allows to easily pick out the best determined atom set from a set of redundant conformers e STDOUT is now flushed regularly to make monitoring easier Version 8 0 2c 1 Mar 2002 e A warning is now given if a pdb file contains B values that are smaller or equal 0 0 such B values would mess up the error estimation e The manual is again available in pdf format e The plotting if DD matrices for multi chain problems is still not perfect loads of book keeping to do but at least does not hang anymore e The program will now give an estimate on the memory it will use If this estimate is close to the physical RAM of your computer try to use a mashine with more memory to avoid swapping Version 8 0 2 1 Feb 2002 e Rewrote documentation to reflect zillions of changes e Major improvements on RASMOL MOLSCRIPT and LSQKAB scripts generation e Substantial cleanup on the log file Version 8 0 1g 5 Jun 2001 e Fixed some problems with MOLSCRIPT output of the rigid b
3. e aset_egen now has a keyword esd_blim to select the lowest accepts B value e if secondary structure is plotted now the data for this come from a reference set The number of this set defaults to the first set selected but can be changed using the setr keyword of ddm e sele now works properly with chainid e aset_write can now write selected sets of atoms e The program can now hold 100 atom sets e Some cleanup on eps output files e Started a chapter on tips amp tricks Version 3 0 1e 9 Jan 2001 e Chorismate Mutase was included as an example into the user s manual e Hopefully the use of Equation 26 or 27 from Cruickshank s paper 2 is now somewhat clearer e When reading coordinates from a file using the aset_read command atoms can now be selected directly i e if you only want CA atoms from a file called fname pdb use aset_read ifile fname pdb sele name CA Version 6 0 1d 14 Nov 2000 e A number of things concerning the refinement are now extracted from the pdb file and passed on to the aset_egen command I am not sure how to determine a sensible number of paramters to be used in Cruickshank s equation 26 so at the moment number of parameters is set to 0 causing Cruickshank eq 27 the one based on Rfree to be used e fixed mistake in URL given in program startup message e fixed bug concerning y ticks Version 3 0 1c 1 Nov 2000 e The manual was updated e If the keyword check in
4. vy2 350 xticks bottom ddm seta 2 setb 3 vx1 170 vy1 490 vx2 240 vy2 560 xticks off yticks off ddm seta 2 setb 4 vy1 420 vy2 490 ddm seta 2 setb 5 vy1 350 vy2 420 now the same procedure for the equivalent error scaled matrices 15 seta 1 setb 2 lolim 3 0 hilim 5 0 vx1 170 vy1 630 vx2 240 vy2 700 type both esd_scaled on limit 150 ticks off comments off xticks off yticks off legend on legendx 340 legendy 200 legendw 180 J ddm seta 1 setb 3 vx1 240 vx2 310 legend off ddm seta 1 setb 4 vx1 310 vx2 380 ddm seta 1 setb 5 vx1 380 vx2 450 ddm seta 1 setb 6 vx1 450 vx2 520 ddm seta 2 setb 3 vx1 240 vy1 560 vx2 310 vy2 630 ddm seta 2 setb 4 vx1 310 vx2 380 ddm seta 2 setb 5 vx1 380 vx2 450 ddm seta 5 setb 6 vx1 450 vy1 350 vx2 520 vy2 420 ddm seta 1 setb 1 vx1 100 vy1 630 vx2 170 vy2 700 ticks auto yticks left ddm seta 2 setb 2 vx1 170 vy1 560 vx2 240 vy2 630 yticks off ddm seta 3 setb 3 vx1 240 vy1 490 vx2 310 vy2 560 ddm seta 4 setb 4 vx1 310 vy1 420 vx2 380 vy2 490 ddm seta 5 setb 5 vx1 380 vy1 350 vx2 450 vy2 420 ddm seta 6 setb 6 vx1 450 vy1 280 vx2 520 vy2 350 xticks bottom and we are finished Not so bad stop The figure produced Figure 3 4 represents a huge amount of information on a single A4 page Actually to be honest I had to modify the header of the postscript file to get a usable figure Old postscript head
5. 2 D W J Cruickshank Remarks about protein structure precision Acta Cryst D55 583 601 1999 3 C C Hyde and E W Miles The Tryptophan Synthase Multienzym Complex Explor ing the Structure Function Relationships with X ray Crystallography and Mutagenesis Bio Technology 8 27 32 1990 4 W Kabsch A discussion of the solution for the best rotation to relate two sets of vectors Acta Cryst A34 827 828 1978 5 T R Schneider Objective comparison of protein structures error scaled difference distance matrices Acta Cryst D56 714 721 2000 6 T R Schneider A genetic algorithm for the identification of conformationally invariant regions in protein molecules Acta Cryst D58 195 208 2002 7 T R Schneider E Gerhardt M Lee P Lian K S Anderson and Schlichting I Loop closure and intersubunit communication in tryptophan synthase Biochem 37 5394 5406 1998 27
6. Right now however you would need to allign the molecules externally and then compare the matching segments Let s say you have molecules A and B and you have matches like A10 A20 lt gt B15 B25 A25 A30 lt gt B42 B47 A50 A70 lt gt B48 B68 Then you could do selections like aset_sele tset 1 sele resi in 10 20 25 30 50 70 aset_sele tset 2 sele resi in 15 25 42 47 48 68 and run a normal ddm analysis using the keyword check loose to allow non identical residue names for atoms being compared ddm check loose seta 1 setb 2 5 0 3 How do I avoid total confusion when colouring superimposed models in MOLSCRIPT A good strategy is to first extract only the pieces of the moved files that you really want read tmp pdbihvy ent copy mol0 require in chain A and in amino acids delete tmp 21 read tmp pdbihw3_mr pdb copy moll require in molecule tmp in amino acids and in chain A delete tmp For colouring first colour everything as flexible and then mark the rigid blocks with another colour set residuecolour molecule mol rgb 1 0 0 35 0 35 flexible set residuecolour from 61 to 94 rgb 0 5 0 5 1 0 rigid set residuecolour from 99 to 104 rgb 0 5 0 5 1 0 rigid set residuecolour from 133 to 147 rgb 0 5 0 5 1 0 rigid The selection for the rigid pieces will only adress existing residues as this is in fact part of the algorithm an atom has to be existing in all models in order to
7. sets in the ddm command in the above script ddm setl 1 3 lt was setl 1 5 esd_scaled on lolim 2 0 hilim 5 0 ssplot on rbplot on J The main result of the analysis is gt The following residues form a conformationally invariant region A4 A12 A47 A226 A232 A328 Now you can use the corresponding Cg atoms in your favourite fitting program You can also try to use the LSQKAB script that ESCET has generated for you t_lsqkab csh As for the previous examples there are some premade RASMOL and MOLSCRIPT scripts Running molscript r in t_molscript mol render jpeg gt aatase_ddm2 jpg will give you a plot like the one in Figure 3 2 Figure 3 2 MOLSCRIPT figure of a molecule of Aspartate Aminotransferase with rigid parts mapped onto the molecule in blue flexible parts in red For this figure the script t_molscript mol generated by ESCET the only editing done concerned the orientation matrix 3 3 Tryptophan Synthase Tryptophan synthase catalyses the last two reactions in the biosynthesis of tryptophan the cleav age of indole 3 glycerol phosphate IGP to indole and glyceraldehyde 3 phosphate a reaction and the subsequent condensation of indole with serine to form tryptophan reaction 3 The 11 reactions take place at two active centers which are separated by a distance of more than 25 A but nevertheless are precisely synchronized 1 In a study aimed at the understanding of the interacti
8. the ddm command is set to auto the program will try to find a consistent set of atoms for the subsequent calculations all by itself This works in many cases but not always If it does not work the selection can still be done by hand using check loose or check strict I will try to make the algorithm more robust in the future so please send me examples were the automatic selection does not work e Several atom sets can now be simultaneously selected by the set1 keyword of various commands The most important application of this is for difference distance matrices if you select a list of atom set via the setl keyword all pairwise difference distances will be printed Each matrix will be put onto a separate sheet producing a sort of book See example on page 13 25 e Atoms can now be selected based on their element or number in their atom set This is very handy if you want to exclude hydrogens see example on page e g the following script will get rid of all hydrogen atoms found in a pdb file aset_read ifile test pdb aset_sele sele element lt gt H aset_write ofile test_without_hydrogens pdb e A much easier mechanism for selecting stretches of residues has been implemented e g selecting residues 19 to 25 36 to 42 45 and 56 to 58 can now be done in a statement of the form sele resi in 19 25 36 42 45 56 58 e Some facilities to work with multiple models from NMR spectroscopy have been included The informa
9. type lower use only lower half of matrix to display result comments off do not put comments 12 pstype eps make an eps file psfname trps_ddmi eps name of eps file title DD matrices for TRPS put a title ssplot on put secondary structure scheme J now display normal matrix seta 1 setb 2 atom sets to use esd_scaled off this time no error scaling lolim 0 3 hilim 2 0 display limits type upper use only upper half for display plot a black diagonal ddm seta 1 setb 2 type diag stop This script produces the plot shown in Figure 3 3 3 4 Mersacidin Mersacidin is a polypeptide antibiotic containing 20 amino acids that crystallizes with six molecules in the asymmetric unit The structure was solved and refined against merohedrally twinned data to 1 06 resolution as described in 5 The conformations of the six molecules are similar to each other with a mean rmsd for all 15 possible pairwise least squares superpositions using all C atoms of 0 83 A as calculated by LSQKAB 4 Analysis of the superimposed molecules to identify rigid and flexible regions was inconclusive For six molecules there are 6 x 6 6 2 15 possible pairwise comparisons The analysis of the corresponding 15 difference distance matrices allowed the indentification of rigid and flexible parts of the molecule The analysis is described in 5 For the six atom sets corresponding to the six molecules all
10. ESCET Version 0 3 User Manual Thomas R Schneider January 24 2003 Contents 1 Introduction 2 2 Command Scripts and Data Structure 3 Dl ATOM SOUS ts Boe teh A asl ete Ee en a ee a eB 3 QED ATOMS lt 8 NN 4 2 3 ReSIDUGS anses ga A ee ep a Bae a es i 4 3 Identification of the rigid part of a protein 6 Sil lt Ghorisiriat Mutase so 4 A ee ae ROY ee ses OE Y 7 3 2 Asparte Aminotransferase 2 2 2 a 9 3 3 Tryptophan Synthase 0 e 11 34 Mersacidin ic es S avec Sere Be A eee Eee Eee ee 13 3 5 Comparing models from non identical but very homologeous molecules 16 4 Utilities 18 Al Juggling POB 35 a 22 a OL A Ee ee 18 ADP UMD it aes do A Heals Mees di 19 5 Tips and Tricks 20 6 Release Notes 23 Chapter 1 Introduction ESCET is ascript driven program to analyse and compare three dimensional protein structures The current P test version is mostly for calculating and displaying error scaled difference distance matrices Starting with version 0 2 an automatic algorithm to interpret difference distance matrices has been added 6 This document contains a short introduction to the program The easiest way to get started is to look through the different scenarios discussed in chapter 3 and use one of the examples scripts Running the command escet h from the UNIX command line will create an HTML file called escet_ref html that can be used as a reference manual An ESCET script file can be exe
11. OINT presentation All difference distance matrices are plotted in POSTSCRIPT format This causes Problems when the plots are included into POWERPOINT presentations i e the plots are not visible on non POSTSCRIPT devices I do not know of a program that really converts well between POSTSCRIPT and for example wmf Windows Meta Format The solution is to write the matrix that you want in eps format and then read it into the program GIMP http www gimp org When loading the file you can choose the resolution I found that 300 dpi is usually good enough If the resolution is too low you will see strange interference pattern in the matrix Then save the image in tif format and read it into xv From Xv you can then save the image in gif format gif is very good at storing plots with lots of straight edges in a very compact form This detour is necessary because GIMP does not support gif anymore and XV is not very good at converting POSTSCRIPT to a pixel format Why does the program gets confused by atom names used for my co factor Sometimes atoms of co factors have rather strange names One examples is NADP The pdb says about this at the following location http www rcsb org pdb docs format pdbguide2 2 guide2 2_frame html in large het groups it sometimes is not possible to follow the convention of having the first two characters be the chemical symbol and still use atom names that are meaningful to users A example is nicotinamide ad
12. ayed for comparison For more on this analysis see 5 Mersacidin Section 3 4 How to display 36 matrices for two different ways of comparing 6 NCS related molecules at once For a change estimated standard deviations are available from a SHELXL lst file 3 1 Chorismat Mutase Comparison of three NCS related molecules Here is the script to read the data divide the atoms into NCS related copies and run the analysis aset_read ifile 1DBF pdb sele name CA Generate standard uncertainties based on the information found in the pdb file As the completeness of the data was not present in the pdb file so the value was put in manually aset_egen sset 0 esd_model dpiu cpl 92 0 divide the structure into three independent sets of atoms i a stn Sa a Gs Sg em a pS a as a ct a a a Se ne aset_sele tset 1 sele chainid A aset_sele tset 2 sele chainid B aset_sele tset 3 sele chainid C calculate an plot the error scaled difference distance matrices for all pairs of molecules setl 1 3 list of atom sets to look at esd_scaled on use error scaling lolim 5 0 hilim 10 0 limit for matrix xtint 50 ticks suitable for publication ticksfontsize 14 font suitable for Acta after shrinking ssplot on plot secondary structure rb_plot on plot rigid body description pstype ps produce an ps file on output psfname cmut_ddm_5 0 ps filename for graphics finito stop T
13. condary structure elements If a pdb file is read the program will try to figure out the secondary structure assignment from the HELIX and SHEET records If you are reading the data from a non pdb file a pdb file without the HELIX and SHEET records or if you are not happy with the assignment read you can modify the atom property ssid using the aset_amod command The two commands aset_amod set 1 ssid H sele resi in 3 20 34 40 aset_amod set 1 ssid E sele resi in 24 30 44 50 will modify the atoms in set 1 such that residues 3 to 20 and 34 to 40 will become a helical and residues 24 to 30 and 44 to 50 will become 8 sheet Chapter 3 Identification of the rigid part of a protein Please try to roughly understand the paper on the sub ject 6 before trying to run the program As it stands now the analysis of a set of conformers will proceed as follows Read a bunch of models from one in the case of NCS related molecules or several coor dinate files gt aset_read command Select the atoms that will be analysed Usually these will be C atoms In complicated cases one can also restrict selections to chainids and residue ranges etc to obtain con sistent sets of atoms the program tries to figure out the largest consistent set itself but sometimes needs a bit of help aset_select command In simple cases the selection can be done directly in the aset_read command If no standard
14. cuted from the UNIX command line using a command of the following form escet lt myscript inp gt myscript log amp Diagnostic output will then end up in myscript log graphical output will be dumped to a postscript file Scripts to look at the output using RASMOL and or MOLSCRIPT are put into files called t_ras ras and t_molscript mol respectively Currently the program is only available as an executable for the Linux operating system If you find the program useful please cite the following paper Schneider TR A genetic algo rithm for the identification of conformationally invariant regions in protein molecules Acta Cryst D58 195 208 2002 Thank you Please report all problems to me email trsCshelx uni ac gwdg de The program and its documentation are copyright Thomas R Schneider 2000 02 I would like thank the testers in particular Karl Edman from Uppsala University for putting up with a semi functional version of the program and for making lots of useful suggestions Chapter 2 Command Scripts and Data Structure The program stores data pertaining to atoms in atom sets An atom set can contain an almost unlimited number of atoms the number of sets is currently limited to 100 Atom sets can be manipulated and analysed using a large number of commands The commands are normally put in a command script which is then run from a UNIX shell A typical command script has the form ESCET script for making a B
15. enine dinucleotide atom names begin with an A or N depending on which portion of the molecule they appear in e g AC6 or NC6 AN1 or NN1 Why does LSQKAB not do what I want 1 Make sure that the FIT command is always before the MATCH command 2 Make sure that you are not using the same file for REFCRD and WORKCD 3 Make sure that there is no wrongly formatted metal atoms in the pdb file A typical error message for this is fmt read unexpected character 20 apparent state internal I 0 last format 6X 15 11X 14 lately reading sequential formatted internal 10 Abort How do I use RASMOL There is a very good Quick REference Card at http info bio cmu edu Courses BiochemMols RasFrames REFCARD PDF Another website for RASMOL is at http www umass edu microbio rasmol An overview of a lot of RAS MOL related websites can be found at http www rasmol org After I start ESCET my computer starts swapping Why As it stands the program is not really optimized in terms of memory consumption A matrix for 2000 x 2000 atoms will take 2000 x 2000 x 4 Bytes float 16 MBytes of memory Comparing 6 such models will give 16 matrices i e roughly 256 MBytes of memory are necessary to run such a problem But hey memory is cheap How can I run ESCET on homologous i e non identical molecules models Extending ESCET to work on homologous structures is one of the things I plan to include in the future
16. er PS Adobe 3 0 EPSF 3 0 BoundingBox 50 150 220 680 Modified header PS Adobe 3 0 EPSF 3 0 BoundingBox 50 150 550 750 3 5 Comparing models from non identical but very homolo geous molecules Ran out of steam sorry 16 101 110 120 201 210 220 301 310 320 401 410 420 501 510 520 601 610 620 W 7 E 3 2 0A 0 3 0 3 2 0 5 0 8 0 3005 00 Figure 3 4 Difference distance matrices and error scaled difference distance matrices for the six molecules of mersacidin In the lower left triangle ordinary difference distance matrices for all pairs of NCS copies are shown The color coding is according to the bar on the lower left all changes in distances smaller than 0 3 are shown as gray differences in distances between 0 3 and 2 0 are shown using a color gradient where red stands for expansion and blue for contraction light colors represent small changes dark colors large changes all differences larger than 2 0 are shown as full blue and full red respectively The blocks in the upper right triangle show the error scaled difference distance matrices for all pairs of molecules Here all differences lower than 3 0 times o Af are mapped to gray Changes greater than 3 0 and smaller than 5 0 times o Af are colourcoded using a scheme analogous to the one used for ordinary difference distance matrices 17 Chapter 4 Utilities This chapter contains a loose agglomerate of ESCET scripts that are u
17. factor plot read information about atoms from pdb file 21zt pdb into atom set number one aset_read tset 0 ifile 2lzt pdb select atoms in residues 5 to 100 Use atom set number 0 as the source and atom set number 1 as the target aset_sele sset 0 tset 1 sele resi in 5 100 make a plot of the property bfac of the CA atoms vs residue number for the atoms found in sset 1 rp_plot sset 1 prop bfac aver ca Some output will be printed to the screen graphical output will be dumped to a POSTSCRIPT file Please not that the rp_plot command at the moment only works for proteins and not for RNA DNA but I am trying to fix this 2 1 Atom sets Atom sets represent the information that can be found for example in a pdb file They are basically a collection of atoms with some general information such as name of the protein or the crystallographic unit cell found in the pdb file attached Information about atoms can be read from from various file formats Currently supported formats are pdb SHELXL ins res SHELXL lst As much information as possible is extracted from the respective files e g if an lst file contains s u s these are stored Once atoms have been read sets can be modified by applying selection criteria command aset_select keyword sele All commands related to sets of atoms have a syntax of the form aset_ and are described in the reference manual 2 2 Atoms Atoms are the eleme
18. have a chance to be recognized as rigid 5 0 4 How much memory will my ESCET job require The necessary memory in units of Bytes can be calculated via natom x natom x nmodel x nmodel 2 nmodel x 8Bytes where natom is the number of atoms and nmodel the number of models 22 Chapter 6 Release Notes Known Bugs e For multiple chain analyses the rigid body display on the bottom of the DD matrices is sometimes messed up However the calculations and the RASMOL and MOLSCRIPT files are correct Version 0 3 27 Jan 2002 e Loads of small fixes Version 6 0 2f 27 Jun 2002 e The Bounding Box for encapsulated postscript can now be set explicitly using bx1 by1 bx2 bx1 keywords of graphics related commands e Keywords xtdel1 and xtdel2 can be used to specify up to two tickmarks that will be explicitly deleted if e g if tickmarks overlap on the lower or upper end of a scale e Several problems with tickmarks for multi chain plots have been fixed e title and frametitle are now separate concepts frametitle is the title that will be put into the frame of a plot title is the title that will be put on top of the actual figure Use keywords title and frametitle to set the values If several plots are overlayed in one figure the titles will be overlayed as well Setting title should help in such cases For rp_plot the defaults are title frametitle something invented by the program Version 0 2e 17 Apr 2002
19. metimes the script even works without editing Then simply run it from the command line after changing there permissions to executable of course chmod x t_lsqkab csh WARNING All the scripts that start with t_ will be overwritten relentlessly by subsequent runs of ESCET in the same directory So if you want to safe these scripts rename them In the first run lolim e 50 was used as a lower limit for significant changes To rerun the whole thing with a different setting of e 20 all we have to do is to change the lolim parameter of the ddm keyword and rerun the program The bit that is interesting in the log file can be found by searching the log file for the string gt conformationally invariant region Here the program tells us what it has identified as conformationally invariant gt The following residues form a conformationally invariant region A3 A13 A48 A52 A86 A95 There is some more useful stuff in that part of the file have a look around 3 2 Asparte Aminotransferase Here we read five models from five different files As the pdb files are a bit of a mess we have to put some of the numbers necessary for the calculation of uncertainties by hand Read the models keep only CA atoms and take only the first chain if there are several chains aset_read tset 1 ifile 7AAT pdb sele name CA and chainid A aset_read tset 2 ifile 1TAR pdb sele name CA and chainid
20. ntary unit of the program An atom has a number of properties that can be used and modified Examples of such properties are x y z coordinates atom name chain identifier chemical element B factor etc pp 2 3 Residues In the framework of ESCET a residue is a set of atoms that share the same chain identifier and the same residue number To count a residue must have at least one atom named CA If properties of entire residues e g the average B factor of the sidechain atoms have to be stored the information is kept with the respective C atom 2 3 1 Secondary structure information A typical property of a residue is the type of secondary structure element it is located in This information is stored as a single letter in the atom property ssid for secondary structure iden tifier of the respective C atom The single letter code corresponds to the Kabsch amp Sander notation as for example used in PROCHECK The codes and their meanings are given in Ta ble 2 3 1 code meaning B residue in isolated beta bridge E extended strand participates in beta ladder G 3 helix 3 10 helix HP 4 helix alpha helix TP 5 helix pi helix S bend I hydrogen bonded turn e extension of beta strand extension of 3 10 helix h extension of alpha helix t used in PROCHECK but I don t know what it means no secondary structure Table 2 1 Identifiers used for se
21. o run this script type gt escet lt cmut_ddm_5 0 inp gt cmut_ddm_5 0 out This will dump the difference distance matrices into a file called cmut_ddm_5 0 ps You can look at this file with GHOSTVIEW if your are interested Otherwise you can check out the log file cmut_ddm_5 0 out Most interesting is probably the graphical representation of the results files called t_ras ras and t molscript mol have been created that can be used to show the results using RASMOL and MOLSCRIPT respectively Typing rasmol script t_ras ras will bring up a nice little RASMOL window displaying your molecule with the conformationally invariant atoms in blue and the flexible ones in red see Figure 3 1 a Figure 3 1 a RASMOL display of a molecule of Chorismate Mutase with rigid parts mapped onto the molecule in blue flexible parts in red b The same with MOLSCRIPT Typing molscript r lt t_molscript mol render jpeg gt t_molscript jpg will produce a rendered molscript plot See Figure 3 1 b Of course you may need to put the correct coordinate transformation into the MOLSCRIPT input file first If you have the opengl version of MOLSCRIPT this is very easy There is also a script to run the CCP4 program LSQKAB on the different structures t_lsqkab csh As such a superposition can be quite complicated the script is also a bit com plicated and may need some editing I have tried to put some useful comments into this script But so
22. ody finder e Argument dd_plot off in keyword ddm will switch off the postscript output Version 0 1f 25 May 2001 e The conformationally invariant part of a molecule can now be automatically determined using a ge netic algorithm Right now no parameters are accessible from the interface changing parameters still has to be done in the source But using rb find on in the ddm command will run the algorithm using a reasonable set of default parameters Setting rb_plot on and ss_plot on will put the information also into the plotted difference distance matrices rigid parts are marked as dark gray flexible as light gray The program also dumps a bunch of files to use as input to MOLSCRIPT RASMOL and LSQKAB I will work hard on documenting these promised molscript gl lt t_molscript mol will give a pretty picture in many cases blue for rigid red for flexible If it doesn t work it will at least be a good template 24 If more than two atom sets were selected using the setl keyword in the ddm command the auto matic interpretation will take all models into account automatically e In previous version the Cruickshank error estimate was calculated deriving some of the input numbers from the current atom set If the calculation was done after removing some atoms or only retaining the CA s this could give wrong results The program now remembers the numbers from the original pdb file and uses those as default values
23. on between the two active sites crystal structures of the enzyme in complex with the substrate analogue 5 fluoroindole propanol phosphate TRPS pdb entry 1A50 and in complex with both F IPP and L serine TRPSI where A A stands for the amino acrylate that is formed at the P site under the experimental conditions chosen pdb entry 1A5S were determined 7 The complete difference distance analysis is described in 5 read the structure with IPP bound and select the bits that we want aset_read ifile 1A50 pdb aset_select tset 1 sele name CA and chainid B and resi in 3 389 a read the structure with IPP and AMAC bound and select the bits that we want aset_read ifile 1A5S pdb aset_select tset 2 sele name CA and chainid B and resi in 3 389 J generate coordinate uncertainties for the first structure aset_egen sset 1 atom set 1 esd_model dpiu use DPI plus linear B scaling ni 5191 number of atoms nobs 31627 number of observables cp1 95 6 completeness r ree 22 1 free R value dmin 2 29 maximum resolution of x ray data Je the same for the second structure aset_egen sset 2 esd_model dpiu ni 5148 nobs 30327 cp1 93 8 rfree 24 7 dmin 2 30 Ja first display error scaled matrix seta 1 setb 2 atom sets to use esd_scaled on use error scaling rbfind off don t run genetic algorithm lolim 1 5 hilim 5 0 display limits
24. or to being displayed Below the matrix secondary structure elements are shown schematically open boxes stand for helices filled boxes for beta sheets automatically produce all error scaled difference distance matrices ddm setl 1 6 work on all six sets check loose loose consistency check 14 esd_scaled on esd scaling is used lolim 3 0 hilim 5 0 low and high limit for DD matrix psfname ddm_mers_all ps postscript output file stop Following is a script that will produce something similar to Figure 2 in the paper 5 i e all difference distance matrices in one plot The script is somewhat envolved I hope I will automate this in the future but I include it here in case you are facing a similar problem Reading coordinates etc as in ddm_mers_all inp put the very first normal difference distance matrix ddm check loose seta 1 setb 2 lolim 0 3 hilim 2 0 vx1 100 vy1 560 vx2 170 vy2 630 type both esd_scaled off limit 150 ticks auto comments 0ff xticks off ticksfontsize 8 0 legend on legendx 100 legendy 200 legendw 180 pstype eps psfname ddm_mers eps J and the other fourteen changing the viewport position as we go along We also need to worry about putting and not putting ticks ddm seta 1 setb 3 vy1 490 vy2 560 ticks auto xticks off legend off ddm seta 1 setb 4 vy1 420 vy2 490 ddm seta 1 setb 5 vy1 350 vy2 420 ddm seta 1 setb 6 vy1 280
25. possible pairwise difference distance matrices can be plotted on 15 pages of paper using the following script read coordinates and errors from SHELXL list file keep only CA atoms ch ce ao ae Se e e aset_read ifile mers_fin_ls lst sele name CA prepare six atom sets one for each molecule 1 e sr o o ee aset_select tset 1 sele name CA and resi in 101 120 aset_select tset 2 sele name CA and resi in 201 220 aset_select tset 3 sele name CA and resi in 301 320 aset_select tset 4 sele name CA and resi in 401 420 aset_select tset 5 sele name CA and resi in 501 520 aset_select tset 6 sele name CA and resi in 601 620 13 50 100 COC eet HHH 150 200 Tro Poe HHHH H Ht H H H Poet oo oe 250 300 Pee ERA 99400000 rot 5 00 1 56 150 5 00 2 0 0 3A 03 204 Figure 3 3 Difference distance matrix between the structures of tryptophan synthase in complex with F IPP TRPS and in complex with F IPP and amino acrylate TRPSIP In the lower left half the error scaled difference distance matrix is displayed using upper and lower cutoffs of 1 5 and 5 00 respectively In the upper right half the ordinary difference matrix is displayed using a lower cutoff of 0 3 A corresponding to approximately the 1c level as determined by the o4 method For scaling an upper cutoff of 2 0 A has been employed Both matrices underwent 3x3 binning pri
26. see T R Schneider Acta Cryst D58 in the press 2002 TAAT 1TAR 1AMA 1TAS 1TAT 7AAT 100 0 73 3 76 3 82 0 1TAR 81 9 84 5 86 8 1AMA 100 0 100 0x 1TAS 100 0x 1TAT The above table can be used to decide which conformers are redundant and should not be included in the rigid body analysis in order to not overweight such conformers If for a group of conformers all matrix elements are larger than 98 0 a good way to continue is to only include the conformer with the lowest average esd into the subsequent analysis Here for the groups of 7AAT 1TAR and LAMA 1TAS 1TAT the matrix elements indicate that the respective conformers are the same within experimental error To find the most precise representative of each group we can consult another table Some statistics for atomlists to be analysed gt AL 1 lt esd gt 0 107 0 048 from 0 030 to 0 502 7AAT pdb gt 401 sel gt AL 2 lt esd gt 0 326 0 048 from 0 222 to 0 550 1TAR pdb gt 401 sel gt AL 3 lt esd gt 0 252 0 149 from 0 041 to 1 460 1AMA pdb gt 402 sel gt AL 4 lt esd gt 0 299 0 194 from 0 041 to 2 057 1TAS pdb gt 402 sel gt AL 5 lt esd gt 0 403 0 299 from 0 051 to 2 567 1TAT pdb gt 401 sel 10 So we should continue using 7AAT and 1AMA as the best representative of each of the groups To run the analysis for those two models only all we have to do is to change the atoms
27. seful for all kinds of things 4 1 Juggling PDB files 4 1 1 Splitting a pdb file into separate chain id s ESCET script epi_split inp read coordinates of CA atoms from a pdb file prepare ten atom sets one for each molecule ce ee ee a ee ee ee eee aset_select tset 1 sele chainid A aset_select tset 2 sele chainid B aset_select tset 3 sele chainid C aset_select tset 10 sele chainid J now write the all into different files aset_write sset 1 ofile t_A pdb aset_write sset 2 ofile t_B pdb aset_write sset 3 ofile t_C pdb aset_write sset 10 ofile t_J pdb stop 18 4 2 Stuff 4 2 1 Change residue names If you need to change the residue names in a pdb file from 3 letter code to 1 letter code you can use the following commands in this case for atom set number 2 which happens to be a piece of RNA aset_amod set 2 rtype G sele resn GUA aset_amod set 2 rtype U sele resn URI aset_amod set 2 rtype A sele resn ADE aset_amod set 2 rtype C sele resn CYT 4 2 2 Compare two models The aset_comp command will try to do a very simple comparison of two atom sets WARN ING this is very a test aset_read tset 0 ifile coord1 pdb aset_read tset 1 ifile coord2 pdb sele element lt gt H aset_comp seta 0 setb 1 stop 19 Chapter 5 Tips and Tricks How do I put a difference distance matrix into a POWERP
28. tion about rmsd s from the NMR ensemble is translated into positional esd s using the aset_egen command Two models are available esd_model rmsd simply uses the rmsd as the coordinate uncertainty and esd_model rmsd2 uses the square of the rmsd as the coordinate uncer tainty In both cases the uncertainty is multiplied by the number given via the keyword esd_fac to allow to put the coordinate error on some pseudo absolute scale This facility is useful if you want to compare NMR structures and X ray structures See under the aset_egen command in the reference manual e A schematic representation of secondary structure elements can now be included into difference distance matrix plots Helices are shown as open sheets as filled boxes Secondary structure can be defined using the aset_amod command see reference manual and section 2 3 1 If HELIX and SHEET records are found in a pdb file the information is used The keyword ss_plot of the ddm command is used to trigger plotting of the secondary structure For example see Figure 3 3 e A number of small bugs have been fixed Version 0 1 11 Jun 2000 e This is the very first beta test release of ESCET Please be patient and report all errors problems glitches misunderstanding etc p p to trs at shelx uni ac gwdg de 26 Bibliography 1 K S Anderson E W Miles and K A Johnson Serine Modulates Substrate Channeling in Tryptophan Synthase J Biol Chem 266 8020 8033 1991
29. uncertainties have been read from the coordinate file as is the case most of the time unless you are in the fortunate situation of having a SHELXL lst file from a full matrix inversion available some estimates for the coordinate error have to be generated Normally one would use the B factor scaled DPI explained in 5 aset_egen command The program is pretty clever in extracting the information it needs to calculate the error estimates from the pdb file In case not all the necessary pieces can be found the information can be added by hand see section 3 2 Run the automated error scaled difference distance matrix analysis ddm command Look at the results in the log file or use the automatically generated scripts for LSQKAB MOLSCRIPT and RASMOL to create some more intuitive representations The following examples are discussed Chorismat Mutase Section 3 1 Compare three NCS related copies of Chorismat Mutase at 1 3 A Make figures that can be used for publication in Acta Cryst D For more see 6 Aspartate Aminotransferase Section 3 2 Compare five different models of Aspartate Aminotransferase The models have been refined to different resolutions and some of the information necessary to generate standard uncertainties has to added manually For more see 6 Tryptophan Synthase Section 3 3 Two models of Tryptophan Synthase are compared Both normal and error scaled difference distance matrices are displ
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