The user manual
Contents
1. Nolsq End Example 7 The user wants to explore 2 blocks of 300 trials storing 50 sets for the first and 100 sets for the second the program does not apply the RELAX procedure Window Structure crambin Phase Blocks 2 300 50 300 100 Unrelax End 29 Example 8 The program explores only the Patterson trial associated with the peak number 5 it applies the Direct Space Refinement strategy for medium size structures and stops if the final residual value Rcr is less than the threshold indicated by the user Nowindow Structure conotoxin Phase Ptrial 5 Residual 10 0 Size m End Example 9 The user wants to apply the Patterson procedure to the 25 highest peaks in the SMF map window Structure conotoxin Phase Peaks 25 End Example 10 The user wants to explore only the tangent trial associated with the progressive number 26 and stop the program at cycle 8 of the DLSQ procedure window Structure loganin Phase trial 26 cycle 8 End 30 Example 11 In the following example the user knows a fragment and wants to complete it using the Fourier Least Squares procedure The ascii file azet fra must exist WINDOW STRUCUTRE azet Phase Fragment azet fra Recycle CONTINUE Coordinates are in the file azet fra which contains Cl 02944 72012 08865 Cl 23727 78692 30869 31 References Altomare A Cascarano G Giacovazzo C amp Guagliardi A 1993 J Appl Cryst 26 343 350 A2. at the end of the DSR process by the crystallographic residual factor Rcr if the final value of Rcr is smaller than a given threshold default value 0 25 the program stops otherwise the program explores the next ranked phase set For large size molecules and proteins the least squares are very time consuming furthermore they cannot be applied to non atomic resolution data To recognise the correct solution we have devised a new figure of merit fFOM to be applied at the end of the DSR process It is defined as follows RAT snai x CC all nat RAT initial CC all iF OM x COMB na COMB ia initial where CC is the correlation factor between the Ro and R and COMB CC large 3 CC weak The words al large and weak indicate the complete normalized structure factors set ranked in decreasing order the subset of the largest F s 70 of them and the subset of the weakest ones 30 of Fo s respectively The indexes initial and final indicate that the corresponding FOM values are calculated at the beginning and at the end of the DSR procedure FOM shows some quite interesting new features a COMB is constituted by two contributions the first arising from the large and the second from the weak reflections this last with a weight three times larger than the former This weighting criterion is justified by the fact that the weak reflections are not 11 involved in the p
3. applied to break in the S r map the residual Patterson symmetry the final S r map is inverted to provide phases and weights to start the DSR process The multisolution approach is obtained by using as pivots the highest SMF peaks for each pivot a set of phases is obtained which is ranked by a specific early FOM pFOM defined as fs arfs r dr P e ar pFOM eFOM where S and Sz denote the S map before and after the filtering procedure respectively the numerator integrals are calculated for the top 2 part of the map while the integral at denominator representing the normalization factor is calculated for the top 20 part of the Patterson map It is expected that S maps having more pronounced peaks correspond to good solutions The Direct Space Refinement DSR The DSR procedures see Figs 2 3 are constituted by the following steps Burla Carrozzini Caliandro et al 2003 a s supercycles of electron density modification EDM each constituted by microcycles p g p The default values of s and change with the category of the structure The modification of the electron density map includes powering Refaat amp Woolfson 1993 and the inversion of small negative domains Burla Carrozzini Caliandro et al 2003 New phases and normalized structure factor modules R are obtained by inversion of a small percentage few percents of the electron density map such modules are rescaled by histogram ma
4. structure is used to create file names WINDOW Graphic window is required NOWINDOW Graphic window is suppressed Directives are described below in the sections dedicated to the various routines All commands and directives are in free format between columns 1 80 and are case independent Only the first four characters are significant The keywords can start in any position If the first non blank character is gt or then the record is interpreted as a comment characters following gt or will be ignored Sir2004 preserves intermediate results For example if invariant estimates have been already obtained during a previous run of Sir2004 in a new run the commands INVARIANTS can be omitted Commands can be given in any order under the following conditions first routine used must be DATA if it has not been used in a previous run INVARIANTS routine has no meaning if observed structure factors are not normalized PHASE routine has no meaning if no triplets have been calculated The minimal information needed by Sir2004 is constituted by cell parameters cell content space group symbol reflections 22 Directives and their use Data Routine Only directives marked in red are mandatory CELL a bcafy Cell dimensions a b and c are in Angstrom a B and y in degrees ERRORS esd a esd b esd c esd a esd B esd y Estimated standard deviations for the unit cell dimensions
5. up to 5 atoms per asymmetric unit small up to 80 medium up to 200 large up to 600 Xlarge up to 1000 and XXlarge no upper limit While the same tangent procedure is applied to all the categories the DSR may be accomplished in different ways according to the category it requires increasing computing times moving from Xsmall to XXlarge structures We shortly describe the various tools the user may employ in the phasing process The Tangent procedure In Sir2004 a multisolution approach is used the phasing procedure involves for each trial the application of a single tangent ST procedure Burla Carrozzini Cascarano et al 2003 to Niarge reflections those selected by the normalization process starting from a subset of random phases Baggio et al 1978 Burla et al 1992 Besides triplets also the most reliable negative quartets may be actively used during the phasing process Each relationship is used with its proper weight the concentration parameter of the first representation G for triplets or C for quartets The phasing process of Sir2004 computes an early figure of merit eFOM for each tangent trial only the best trial solutions sorted by eFOM are submitted to the DSR module This phasing strategy allows us to explore numerous trials without paying so much in terms of computing time it thus increases the probability of submitting to the DSR procedure a good set of phases In this way we obtain the following adv
6. without any prior information on the crystal structure or on the molecular envelope A simple solvent model arising from the Babinet s principle is used i e Fom F 1 K exp Bsr where r 2 sin A K is a suitable scale factor in the range 0 7 0 9 and the B parameter is a correction term arising from the larger vibrational motion of the solvent atoms in the range 200 500 in accordance with Tronrud 1997 The expected effects of modifying the observed intensities may be so described a low resolution reflections have F modules too small to influence the phasing process In the practice very low resolution reflections are out of such process on the contrary the corresponding F values much larger then Fol s e g 5 times larger could drive the phasing process along different directions b Low resolution reflections are insensitive to fine structural details but are particularly able to define the location and the envelope of the molecule Accordingly they play a critical role in defining the region of the electron density map selected for performing the transformation 0 in the following steps Phasing Tools According to circumstances the Sir2004 phasing process may apply tangent procedures and or Patterson methods phase extension and refinement are achieved by Direct Space Refinement DSR techniques To preserve its efficiency the program distinguishes different categories of structures Xsmall molecules
7. 2 Birger M J 1959 Vector Space chapter 11 Wiley New York Burzlaff H amp Hountas A 1982 J Appl Cryst 15 464 467 Cascarano G Giacovazzo C amp Guagliardi A 1991 Acta Cryst A47 698 702 Cascarano G Giacovazzo C amp Lui M 1988 a Acta Cryst A44 176 183 Cascarano G Giacovazzo C amp Lui M 1988 b Acta Cryst A44 183 188 Cascarano G Giacovazzo C Burla M C Nunzi A amp Polidori G 1984 Acta Cryst A40 389 394 Cochran W 1955 Acta Cryst 8 473 478 Fan Hai Fu Yao Jia Xing amp Qian Jin Zi 1988 Acta Cryst A44 688 691 Giacovazzo C 1976 Acta Cryst A32 958 966 32 Giacovazzo C 1977 Acta Cryst A33 933 944 Giacovazzo C 1980 Acta Cryst A36 362 372 Giacovazzo C Burla M C amp Cascarano G 1992 Acta Cryst A48 901 906 Hirshfeld F L 1968 Acta Cryst A24 301 311 Leslie A G W 1987 Acta Cryst A43 134 136 Matheus B W 1968 Mol Biol 33 491 497 Pavel ik F 1988 Acta Cryst A44 724 729 Paveltik F Kuchta L amp Sivy J 1992 Acta Cryst A48 791 796 Refaat L S amp Woolfson M M 1993 Acta Cryst D49 367 371 Richardson J W amp Jacobson R A 1987 In Patterson and Pattersons Ed by Glusker J P Patterson B K amp Rossi M pp 310 317 Oxford University Press Sheldrick G M 1992 In Crystallographic Computing 5 Ed by Moras D Podjar
8. Cl weak we wR if RCT peak lt RCT oar and RCT orge lt RCT orge di large wR and wR are the average values calculated for the large and the weak reflections for large Cl weak a given trial Rcr 4 and REP e are the average values of the crystallographic residuals weak obtained over all the trials of the considered block and 01 02 03 are the corresponding standard deviations eFOM is expected to be a maximum for the more promising phase sets those to be processed by DSR procedures The RELAX procedure A systematic error in the ST phasing process as in any other phasing tool may provide well oriented but misplaced molecular fragments eFOM usually ranks such trials among the most promising ones In order to recover the information related to a translated model it is necessary to shift the model to the correct position or equivalently to find the correct phase shift The success has been obtained by the RELAX procedure Burla et al 2002 Its main steps are a the so called Cheshire cell Hirshfeld 1968 is automatically defined The search of a suitable origin translation may be restricted to it b The reflections are expanded in P1 Sheldrick amp Gould 1995 in order to relax symmetry constraints Symmetry related reflections are given values in accordance with the rule Fir F bR h 2xhT c Suitable figures of merit fom and fom2 are automatically computed by the
9. L more the 1000 S 15 M 7 N 6 Heavy atoms Table 4 Default number of DSR iterations versus Nasym RES and Zz ATOMIC RES ATOMIC RES NON ATOMIC RES NON ATOMIC RES Zu gt 20 Za lt 21 Zu gt 20 Zu lt 21 Nasym lt 81 80 lt Nasym lt 201 200 lt Nasym lt 601 5 600 lt Nasym lt 1001 l 4 7 7 Nasym gt 1000 l 4 9 9 20 Commands and their use The input consists of a sequence of comments commands and directives The commands are headed by character and directives must follow the related command Sir2004 recognizes the following commands INITIALIZE Initialize the direct access file to override previous results and data DATA Data input routine INVARIANTS Invariants routine PHASE Phasing routine Direct Space Refinement routine END End of the input file JOB A caption is printed in the output CONTINUE The program runs in default conditions from the last given command up to the end STRUCTURE string This command is used to specify the name of the structure to investigate The program creates the name of some needed files adding the appropriate extension to the structure name The file names are string bin gt direct access file string res gt file in SHELX frmat string plt gt file for graphics 21 If STRUCTURE command is not used the default string STRUCT instead of the name of the
10. SPACEGROUP string String is the symbol or the number of the space group according to International Tables 1974 Blanks are necessary among the terms constituting the space group symbol see examples at the end of this manual CONTENT El n El m El m Unit cell content El is the chemical symbol of atomic type i n is the corresponding number of atoms in the unit cell up to a maximum of 8 atomic types For each chemical element up to Californium Cf Z 98 X ray and electrons scattering factor constants are stored together with information on the atomic number and weight covalent and Van der Waals radii etc in a file see notes on implementation SFACTORS El a bi a2 bo a3 b3 a4 ba Cc Scattering factors for species El If more lines are necessary use character at the end of the line ANOMALOUS El Af Af Values of Af and Af for species El RHOMAX x Maximum value of sin A accepted for reflections to be used In default all the data are accepted RESMAX x Maximum value of resolution in Angstrom accepted for reflections to be used In default all the data are accepted 23 FORMAT string String is the run time format to read reflections Default value for string is 314 2F8 2 RECORD n It specificies the number of reflections per record when n gt 1 REFLECTIONS string Ri Rio R43 R2 R22 R23 R31 R32 R33 String is the name of the reflections file Records have n reflections ea
11. TETS To not calculate negative quartets Default uses them Phase Routine SIZE xs s m l xl xxl To set a suitable solution strategy Default procedure is automatically chosen by the program on the basis of the structural complexity NNEG The program does not actively use negative triplet relationships NNQG The program does not actively use negative quartet relationships BLOCK m nn ma Dil Ni2 The program explores m blocks up to 10 of nj trials and then selects the most promising ni2 up to 300 phase sets those with the higher eFOM score to perform DSR procedures Default values are automatically chosen by the program on the basis of the structural complexity ITERATION n The program performs n up to 25 Direct Space Refinement iteration s TRIAL n The program explores only the tangent trial associated with the progressive number n STRIAL n The program starts from the n tangent trial RELAX The programs applies the RELAX procedure exploring all the phase sets selected for any block of trials UNRELAX The programs does not apply the RELAX procedure PATTERSON The program applies the Patterson procedure NOPATTERSON 25 The program does not apply the Patterson procedure PEAKS n The program applies the Patterson procedure to the n up to 50 highest peaks in the SMF map PTRIAL n The program explores only the Patterson trial associated with the peak number n NOLSQ The program
12. antages 1 to lead to a quick solution those structures for which owing to the good triplet estimates the ST module frequently provides a favourable structural model ii to solve those structures for which the triplet invariant estimates are so bad that a large number of trials are necessary before a good set of phases can be produced by the ST module and subsequently submitted to the DSR procedure iii to solve even those protein structures for which the eFOM ranking is relatively inefficient The early Figure of Merit eFOM eFOM is defined as follows a for small and medium size structures eFOM RAT where Burla et al 2002 CC R RAT R CC pis the correlation coefficient in the range 0 3 1 2 between the R s say R E and the corresponding Sim like coefficients w D 2R R R say R E are the modules of the normalized structure factors available after the inversion of a small percentage 3 5 of the Cc electron density map R is calculated over the 30 of the measured reflections those with the weakest Fo values they are never actively used in the phasing process b for large molecules and proteins eFOM weF OM as defined by Burla et al 2004b wej lt we gt 0 weFOM we lt we gt 02 Wez lt Wez gt 03 where WR ge if R R d R R we if cr weak lt Chica an Charge gt Charge Cl weak l if RCP oak gt Rea We RJ R j ue
13. ative quartets are also generated by combining the psizero triplets relating two reflections with large E and one with E close to zero in pairs those with cross magnitudes smaller than a given threshold are estimated by means of their first representation as described by Giacovazzo 1976 These quartets are actively used in the phasing process Giacovazzo et al 1992 Phase Module Before starting the phasing process the observed E values of the low resolution reflections up to S are modified Burla et al 2000 This action is undertaken since large regions of the unit cell of macromolecular crystals are filled by solvent Since the molecular envelope is usually unknown at this stage low resolution diffraction intensities contain an unpredictable but important solvent contribution Restating more reasonable values for protein diffraction intensities by proper subtraction of the solvent effects may be decisive for the success of the phasing process When the structure of the macromolecule is known at high resolution information about the solvent structure may be obtained by assuming Fo Fp Fs where F is the observed value for the structure factor complex quantity Fp is the protein structure factor calculated from the known coordinates and F is the solvent contribution When the structure is unkown the problem is the reverse the aim is to obtain at low resolution more reasonable F values from the F ones
14. ch with h k l F o F where h k l are integer up to 512 If the orientation matrix Rij is supplied it is immediately applied to reflections and all calculations will be performed using the final orientation The end of reflections is detected using one of the following blank record end of file Negative values of F are allowed negative values of o F are forbidden NOSIGMA To be used when o F values are meaningless or not available FOBS Program assumes h k l F o F F and o F are expected as default FOSQUARED Program assumes h k l F and o F default choice WAVE string Nevertheless the wavelength is not used during the structure solution stage it is necessary for the LSQ refinement its value is also written in CIF file produced by the program Possible values for string are Cu Mo or a numeric value Default wavelength is Mo NREFLECTION n Number of active reflections with largest E values up to 4000 subject to a minimum value of E 1 2 Default number is computed by the program BFACTOR x Isotropic thermal factor if the user wants to supply it The scale factor is assumed equal to 1 ELECTRONS This directive specifies that electron diffraction data will be used Invariants Routine 24 GMIN x Triplets with G lt x are not actively used Default value x 0 3 in any case x gt 0 1 COCHRAN To use the Cochran distribution Po formula is used by default NQUAR
15. does not perform the automatic Diagonal Least Squares calculations CYCLE n The program stops at cycle n of the automatic Diagonal Least Squares calculations RESIDUAL x The program stops if the final crystallographic residual factor Rer is less than the specified value x The default value is 25 This directive is meaningless for macromolecules FRAGMENT string Used to supply a known fragment String is the name of the file in which for each atom are stored the following data Element X Y Z B iso RECYCLE Used to complete a known fragment supplied to Sir2004 CRYSTALS The user wants to produce the output file in CRYSTALS format SHELX format is used by default 26 Examples of input for Sir2004 Example 1 The following example shows the maximum default use of Sir2004 Most of the structures can be solved in this way Diffraction data are in the file crambin hkl in format 314 2F8 2 one reflection per record Data Cell 40 763 18 492 22 333 90 00 90 61 90 00 SpaceGroup P 21 Content C406H776N 1100 131 S 12 Reflections crambin hkl Continue Example 2 In the following example experimental data are stored as F not F using the format 3 314 f10 3 8x f8 2 3 reflections per record Y Window Structure iled Job Isoleucinomycin Initialize Data cell 11 516 15 705 39 310 90 00 90 00 90 00 spacegroup P 212121 content C 240 H 408 N24 O 72 reflections iled hkl record 3 format 3 314
16. e crystallographic residual factor and Rth is its related threshold in default Rth 25 DIRECT SPACE REFINEMENT Automatic DLSQ End Automatic Procedure Automatic DLSQ Netx Set 17 Fig 3 Flow diagram of the DSR procedure for macromolecules Next Iteration Last Iteration EDM HAFR LSQH iii oi 18 Table 1 Small and medium size molecules default values of n m for each block of trial solutions 1 Block 2 Block 3 Block Nasym lt 81 100 10 100 20 100 30 80 lt Nasym lt 201 300 50 300 100 300 200 Table 2 Large structures default values of n m versus structure complexity and data resolution ATOMIC RES ATOMIC RES NON ATOMIC RES NON ATOMIC RES ANY RES Zy gt 20 12 lt Zy lt 21 Zn gt 20 12 lt Zy lt 21 Zy lt 12 200 lt Nasym lt 601 200 100 400 200 200 100 600 300 2500 300 600 lt Nasym lt 1001 200 100 1200 300 1500 300 2100 300 2500 300 Nasym gt 1000 2500 300 2500 300 2500 300 2500 300 2500 300 19 Table 3 Strategy of DSR procedures as function of structural complexity Size Atomsin a u EDM HAFR LSQH XS up to 5 S 6 M 5 N 7 NO S 6 80 S 6 M 5 N 7 NO M 81 200 S 9 M 5 N 7 NO L 201 600 S 15 M 7 N 6 Heavy atoms XL 601 1000 S 15 M 7 N 6 Heavy atoms XX
17. e current phases Burla Carrozzini Cascarano et al 2003 in the following this technique will be called iteration This iterative process although time consuming allows to solve also resistant molecules i e protein structures diffracting at non atomic resolution 10 The molecular envelope The molecular envelope of the protein Wang 1985 Leslie 1987 is used in Sir2004 as a mask in the density modification step in order to improve its efficiency in solving protein structures Burla Carrozzini Caliandro et al 2003 The protein volume is calculated through the Mathews 1968 formula and the envelope is calculated for each trial solution from the current phases The electron density map is modified by assigning weights equal to 1 0 to pixels belonging to the envelope and weights equal to 0 5 to pixels out of it so tentatively depleting the intensities of the false peaks The map is then inverted and the resulting phases may improve their values The envelope information cannot be used just after the tangent formula when a few low resolution reflections are usually phased and when the mean phase error is normally too large The molecular envelope is thus calculated for the first time after three macrocycles of EDM and then recursively calculated and applied in the following DSR procedures Identification of the correct solution the final Fom fFOM For small medium size structures the correctness of a solution is assessed
18. ections It includes a modified version of the subroutine SYMM Burzlaff amp Hountas 1982 Symmetry operators and information necessary to identify structure invariants estimated in INVARIANTS module are directly derived from the space group symbol Diffraction data are checked in order to merge equivalent reflections to find out systematically absent reflections which are then excluded from the data set and eventually weak reflections not included in the data set Cascarano et al 1991 Diffraction intensities are normalized using the Wilson Method Statistical analysis of intensities is made in order to check the space group correctness to suggest the presence or absence of the inversion centre and to identify the possible presence and type of pseudotranslational symmetry Cascarano et al 1988 a b Fan et al 1988 Possible deviations of displacive type from ideal pseudotranslational symmetry are also detected Niarge reflections those with the largest E values are selected for invariants calculations their maximum number is 4000 Invariants Module Up to 300000 triplets relating the Niarge reflections are stored for active use in the phasing process Negative quartets are generated by combining the psizero triplets relating two reflections with large E and one with E close to zero in pairs those with cross magnitudes smaller than a given threshold are estimated by means of their first representation as described b
19. f10 3 8x f8 2 fobs Continue 27 Example 3 The user wants to supply the value for the isotropic thermal factor and to set the number of strong E value reflections Window Structure ferre Job ferredoxin pdb code 2fdn Initialize Data cell 33 95 33 95 74 82 90 00 90 00 90 00 spacegroup P 43212 content C 1824 H 2744 N 488 O 478 S 128 Fe 64 reflections ferre hkl bfac 3 5 nref 2000 Continue Example 4 In the following example the Cochran formula is applied and all triplets with a concentration parameter greater than 0 2 are actively used in the phasing process as requested by the user The binary file ferre bin must exist Commands or directives following gt or are interpreted as a comment and will be ignored WINDOW STRUCTURE ferre gt INITIALIZE gt DATA gt CELL 33 95 33 95 74 82 90 00 90 00 90 00 SPACEGROUP P 43 21 2 CONTENT C 1824 H 2744 N 488 0478 S 128 Fe 64 gt REFLECTIONS ferre hkl INVARIANTS GMIN 0 2 COCHRAN PHASE END 28 Example 5 The user wants to explore new trials starting from the trial number 132 from the phasing process up to the Fourier Least Squares refinement The graphical interface is not used The binary file iled bin must exist Nowindow Structure iled Phase strial 132 End Example 6 The user wants to explore only trial number 154 The program stops before DLSQ calculations Nowindow Structure loganin Phase trial 154
20. hasing process so that CC weak is like a free index much more reliable than the companion contribution CC arge It is frequently negative for the wrong trials b FOM is the product of three figures of merit every value depends on how each FOM is modified during the DSR process not on the value it assumes at the end of the process The correct solution should be identified by large values of fFOM Sir2004 strategy for ab initio phasing of crystal structures The Sir2004 flow diagram shown in Fig l is a useful guide for understanding the program strategy We note a The tangent formula is particularly efficient for small structures here the role of the DSR is quite marginal For medium size structures the application of the DSR is more important it is often able to drive phases with large errors to their correct values For proteins the tangent procedures are rather inefficient a large set of trials have to be explored before finding the useful one The Patterson techniques recently developed by Burla et al 2004a are frequently able to find the correct solution in few trials provided some heavy atoms are present in the structure In our experience if the solution is not found in the first Patterson derived trials obtained by using the largest SMF peaks as pivots it is unlikely to find the correct solution in the following ones In accordance with such conclusions SIR2004 uses the following strategy 1 by default for small and med
21. ir 504 The user manual Index Sir2004 General Information and Background Sir2004 Main Features Description of Sir2004 Data Module Invariants Module Phase Module Phasing Tools The Tangent procedure The early Figure of Merit eFOM The RELAX procedure The Patterson deconvolution procedure The Direct Space Refinement DSR The molecular envelope Identification of the correct solution the final Fom fFOM Sir2004 Strategy for ab initio phasing of crystal structure Sir2004 Completion and refinement of the structure The least squares refinement When default Sir2004 fails some advices Commands and their use Directives and their use Data Routine Invariants Routine Phase Routine Examples of input for Sir2004 References PB PB pg pg pg pg pg pg pg PB PB pg PB pg pg pg pg PB PB PB pg pg pg pg pg O o NAN Dna a RA A A Q Ww N N N N N N RR RR NC SJ WNW LA WO WO KY WH BR A_N KF O Sir2004 General Information and Background The SIR SEMI INVARIANTS REPRESENTATION package has been developed for solving crystal structures by Direct Methods The REPRESENTATION THEORY proposed by Giacovazzo 1977 1980 allowed the derivation of powerful methods for estimating structure invariants s i and structure seminvariants s s The mathematical approach makes full use of the space group symmetry SIR uses symmetry in a quite general way allowing the estimatio
22. ium size structures only the ST algorithm is used 11 for large structures the phasing process starts with the Patterson procedure except for the following cases absence of heavy atoms Zy lt 11 very large structures more than 1000 atoms in the asymmetric unit characterized both by data resolution worse than 1 2 and by intermediate heavy atoms up to Ca in these cases the ST is directly used Only 30 Patterson phase sets are generated and explored by the DSR module Three blocks of ST trials are subsequently generated The trials in each block let n be their number for the i block are sorted by eFOM and the DSR module is applied only to the subset of best ranked trials let m be their number The values of n and m are automatically settled by the program as a function of the complexity of the structure in accordance with the defaults schematised in Table 1 Default values of n and m for proteins n and m do not change with the block number are shown in Table 2 the data resolution is also taken into account proteins data collected at non atomic resolution are more difficult to phase By means of directives the user can modify the default choices Tables 1 and 2 are useful to guide the user towards sensitive non default values Nasym is the number of non hydrogen atoms in the asymmetric unit Zy is the atomic number of the heaviest species 12 b The DSR module see Figs 2 3 is constituted by cycles of EDM electron density m
23. n and use of s i and s s in all the space groups The present version of the program Sir2004 Burla Caliandro Camalli Carrozzini Cascarano De Caro Giacovazzo Polidori amp Spagna 2004 is designed to solve ab initio structures of different size and complexity up to proteins provided that data resolution is no lower than 1 4 1 5A Data can be collected with X Ray or electron sources There is no limit to the number of reflections and to the number of atoms in the asymmetric unit The maximum value allowed for h Ik I is 512 The maximum number of different atomic species is 8 Sir2004 includes several new features with respect to the previous version Sir2002 Burla Camalli Carrozzini Cascarano Giacovazzo Polidori amp Spagna 2003 New tools are represented by the use of procedures based on Patterson Methods as alternative to the application of the Tangent Formula in order to compute the starting phase set the introduction of suitable figures of merit FOM s in order to recognize the correct trial solution and the application of new algorithms for solving ab initio protein structures also for quasi atomic resolution data i e the use of the molecular envelope mask The range of options available to experienced crystallographers for choosing their own way of solving crystal structures is rather wide However scientists untrained in Direct Methods or people trustful in the SIR default mode often can solve crystal struct
24. ntributions are included in the refinement by allowing the positional parameters to ride on the corresponding parent atom 5 Floating origin is restrained automatically by setting the restrain on the sum of the appropriate coordinates 6 Refinement of the Flack parameter to evaluate the absolute configuration 7 The possibility to impose conditions constraints or additional information restraints The constrained atoms are regularized to an ideal model structure of known geometry i e benzene ring and this rigid body is refined as compact unit assuming three translational parameters and three angles which define its orientation The method used to compute the coordinates of the model follows the approach described by Arnott amp Wonacott 1966 In order to build the internal Cartesian coordinates the program uses the ASCII file Sir2004 gru which contains models described by the Z matrix formalism The following restraints are available bond distances bond angles planarity Fourier least squares hydrogens and restraints tools are accessible through the Graphical User Interface GUN 14 When default Sir2004 fails some advices Sir2004 has developed an automatic strategy to find the correct solution among the various trials In addition the user can adopt several options to choose his own phasing pathway We quote a the value of NREF number of reflections actively used in the phasing process Niarge is fixed by the p
25. ny A D amp Thierry J C pp 145 157 Oxford University Press Sheldrick G M amp Gould R O 1995 Acta Cryst B51 423 431 Spagna R amp Camalli M 1999 J Appl Cryst 32 934 942 Tronrud D E 1997 Methods Enzymol 277B 306 319 Wang B C 1985 Methods Enzymol 115 90 112 33
26. odification HAFR a selected number of large intensity electron density peaks are expressed in terms of the heaviest atomic species and of suitable occupancy factors and LSQH the isotropic displacement parameters of the heavy atoms are refined via a least squares procedure The reader is referred to Burla et al 2002 for details According to the structural complexity the strategy and the algorithms improved in the DSR section are different see Table 3 The number of total DSR iterations default mode is automatically defined by the program and it is shown in Table 4 It is a function of Nasym RES and Zy The user can change the number of the iterations either for saving computing time or for increasing the chances to solve resistant structures The RELAX procedure is performed only on the first ng ST trials the best eFOM ranked where np is set to 3 5 or 20 for small medium and large size molecules respectively This choice is due to two reasons 1 the use of RELAX is time consuming 11 our tests suggest that trials corresponding to well oriented but misplaced molecules are usually characterized by large values of eFOM RELAX is not applied to Patterson phase sets For small and medium size structures the program stops if the final value of Rcr is smaller than a given threshold default value 0 25 while for macromolecules the PHASE module runs until all the trials fixed by default or by the user have been processed A histogram i
27. program The grid point Xj for which fom fom2 is a maximum should define the correct origin translation d Let Xo be the correct origin shift the P1 phased reflections are then modified into P P 27hX in order to turn back to the original space group in an automatic way At this step we have re established the original space group symmetry to fully accomplish this task we have to select unique reflections and to assign suitable phases to them A default run of Sir2004 automatically applies the RELAX procedure only for few best ranked trials but the user can modify this choice The Patterson deconvolution procedure Procedures alternative to the application of the tangent formula are the Patterson based methods B rger 1959 Richardson amp Jacobson 1987 Sheldrick 1992 Sir2004 uses the approach described by Burla et al 2004a which can be summarized as follows a b c d the superposition minimum function Pavel ik 1988 Pavel ik et al 1992 is calculated by combining all the independent Harker domains of the Patterson map whose number is denoted by m as follows SMF r Min P r C r s l where r C r is a Harker vector corresponding to the s symmetry operator and P denotes the Patterson map the minimum superposition function is obtained by S r Min P r r SMF 1 where r is the position of a pivot peak selected by the program in the SMF map filtering algorithms are
28. rnott S amp Wonacott A J 1966 Polymer 7 157 166 Baggio R Woolfson M M Declerq J P amp Germain G 1978 Acta Cryst A34 883 892 Burla M C Caliandro R Camalli M Carrozzini B Cascarano G De Caro L Giacovazzo C Polidori G amp Spagna R 2004 submitted Burla M C Caliandro R Carrozzini B Cascarano G De Caro L Giacovazzo C amp Polidori G 2004a J Appl Cryst 37 258 264 Burla M C Caliandro R Carrozzini B Cascarano G De Caro L Giacovazzo C amp Polidori G 2004b J Appl Cryst 37 791 801 Burla M C Camalli M Carrozzini B Cascarano G Giacovazzo C Polidori G amp Spagna R 2000 Acta Cryst A56 451 457 Burla M C Camalli M Carrozzini B Cascarano G Giacovazzo C Polidori G amp Spagna R 2001 J Appl Cryst 34 523 526 Burla M C Camalli M Carrozzini B Cascarano G Giacovazzo C Polidori G amp Spagna R 2003 J Appl Cryst 36 1103 Burla M C Carrozzini B Caliandro R Cascarano G De Caro L Giacovazzo C amp Polidori G 2003 Acta Cryst A59 560 568 Burla M C Carrozzini B Cascarano G De Caro L Giacovazzo C amp Polidori G 2003 Acta Cryst A59 245 249 Burla M C Carrozzini B Cascarano G Giacovazzo C amp Polidori G 2002 Z Kristallogr 217 629 635 Burla M C Cascarano G amp Giacovazzo C 1992 Acta Cryst A48 906 91
29. rogram For some special structures the ratio number of active triplets NREF is too small e g less than 20 Larger values of NREF may improve the phasing procedure b Because of a faulty data collection strategy often weak reflections may not be included in diffraction data This lack of information influences both the normalization process scale and overall thermal factors are affected by systematic errors the experimental E distribution is often non centric even when the crystal structure is centrosymmetric and the estimation of invariants in particular a reduced number of negative triplets via Pio formula and of negative quartets is calculated Success in the structure solution may be obtained if weak reflections are also used c High or low resolution reflections may occasionally play a too important role in the first steps of the phasing process Fixing a thermal factor lower or larger than that provided by the normalization routine may successfullly change the phase extension and refinement procedures d Use of the RELAX procedure e An alternative space group should be carefully considered 15 Fig 1 Flow diagram of the SIR2004 program Next Block PHASE Direct Space Refinement yes PATTERSON Direct Space Refinement r End Automatic Procedure 16 Fig 2 Flow diagram of the DSR procedure for small and medium molecules Rcr is th
30. s continuously updated showing the FOM distribution If for a given trial fFOM is outstanding the user can stop the program and submit the selected trial to further analysis i e examination of the electron density map by means of contouring facilities automatic model building using the Final Stage Procedures accessible through the graphical interface If the user is unsatisfied he can launch the PHASE module again starting from the examined trial The above strategy allows to save computing time if the user looks at the FOM histogram 13 Sir2004 Completion and refinement of the structure The least squares refinement As mentioned before for small medium size molecules a preliminary diagonal least squares refinement is automatically performed at the end of the DSR process to recognize the correct solution The least squares module is also suitable for the complete crystal structure refinement Its specific features are 1 the full matrix may be used or any kind of blocks 2 18 weighting schemes are available If the weighting scheme contains adjustable parameters the program refines the values to obtain a good distribution of lt wA gt against F and resolution and the value of the goodness of fit close to one Spagna amp Camalli 1999 3 The program generates constraints for the parameters of atoms on special positions in all space groups 4 Automatic or through wizard generation of hydrogen atoms Their co
31. tching with respect to the distribution of the observed ones R New phases are given a Sim like weight w g RES RES xD 2R R The function g leaves unchanged the weights for the lowest resolution reflections and smoothly increases with sin0 A This feature helps the phasing process for data sets with resolutions worse than 1 2 b When protein data are processed the molecular envelope is calculated from the current phases The electron density map is modified by assigning different weights to pixels falling inside or outside the envelope so tentatively depleting the intensities of the false peaks c DSR includes also cycles of HAFR a selected number of large intensity electron density peaks are expressed in terms of the heaviest atomic species and of suitable occupancy factors and LSQH the isotropic displacement parameters of the heavy atoms are refined via a least squares procedure The reader is referred to Burla et al 2001 for details For small medium size molecules an automatic Diagonal Least Squares refinement DLSQ is applied DLSQ is a procedure which alternates least squares cycles and 2 F Fcl map calculations in order to complete the crystal structure reject false peaks and refine structural parameters Altomare et al 1993 An isotropic diagonal matrix refinement is used H atoms are not involved d The whole DSR procedure could automatically be iterated for the same trial restarting each time from th
32. ures without personal intervention The program is available for Microsoft Windows and Unix SGI Compaq Linux and others platforms see the Notes on the implementation Authors M C Burla R Caliandro M Camalli B Carrozzini G L Cascarano L De Caro C Giacovazzo G Polidori R Spagna O Istituto di Cristallografia CNR Dipartimento di Scienze della Terra Univ di Perugia Support Web site sirmail ic cnr it http www ic cnr it Sir2004 Main Features The program has been designed to require a minimal information in input work automatically reduce the user intervention and facilitate the interaction by means of a friendly graphic interface Description of Sir2004 The main modules of the program are DATA INVARIANTS PHASE The flow diagram of the program is shown in Fig 1 If a graphic interface is active it is possible to interact with the model in order to complete and refine it This option is available only if the number of atoms in the asymmetric unit does not exceed 500 The graphic interface incorporates an online help in order to describe all the features and tools available via graphics Thanks are due to T Pilati Polyhedra representation and to J Gonzalez Platas Contouring tools for their code integrated in Sir2004 Data Module This routine reads the basic crystallographic information like cell parameters space group symbol unit cell content and refl
33. y Giacovazzo 1976 These quartets are actively used in the phasing process Giacovazzo et al 1992 Active triplets may be estimated according to Cochran s distribution 1955 the concentration parameter of the von Mises distribution is then C 2 En Ex En x WN Triplets can also be estimated according to their second representation i e Pio formula as described by Cascarano et al 1984 The concentration parameter of the new von Mises i e of the same form of Cochran s distribution is given by G C 1 q where q is a function positive or negative of all the magnitudes in the second representation of the triplet The G values are rescaled on the C values and the triplets are ranked in decreasing order of G The top ranked relationships represent a better selection of triplets with phase value close to zero than that obtained sorting triplets according to C Triplets estimated with a negative G value represent a sufficiently good selection of relationships close to 180 degrees Positive and negative triplets will be actively used in the phase determination process Giacovazzo et al 1992 The Pio formula is applied as a default to estimate triplets relationships They are ranked in decreasing order of the concentration parameter and actively used only if G is greater than a given threshold Default value is 0 3 If the number of triplets per reflection is too low the program decreases the threshold to a suitable value Neg
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