SHELX C/D/E: Difference between revisions

'''SHELXC''' is designed to provide a simple and fast way of setting up the files for the programs '''SHELXD''' (heavy atom location) and '''SHELXE''' (phasing and density modification) for macromolecular phasing by the MAD, SAD, SIR and SIRAS methods. These three programs may be run in batch mode or called from a GUI such as [[CCP4i]] or (better) [[hkl2map]]. SHELXC is much less versatile than the Bruker AXS XPREP program for this purpose, but if you are sure of the space group and there are no problems with the indexing or twinning and the f’ and f” parts of the scattering factors do not need to be refined, SHELXC should be adequate. SHELXC can read either HKL2000 format .sca files or SHELX .hkl files (F-squared unless the -f switch is used to specify F). To transfer data from CCP4 it is advisable to generate .sca files using 'output unmerged polish' from SCALA or to use the program mtz2sca written by Tim Grüne and supplied with SHELX.  The current version of SHELXC outputs extra useful diagnostic statistics if fed unmerged data. SHELXC, SHELXD and SHELXE are stand-alone executables that do not require environment variables or parameter files etc., so all that is needed to install them is to put them in a directory that is in the ‘path’ (e.g. /usr/local/bin or ~/bin under Linux).

SHELXC reads a filename stem on the command line plus some instructions from 'standard input'. It writes some statistics to 'standard output' and prepares the three files needed to run SHELXD and SHELXE. It can be called from a GUI using a single command line such as:

which would read the instructions from the file t and write the files xx.hkl (h,k,l,I,sig(I) in SHELX HKLF4 format for density modification by SHELXE), xx_fa.ins (cell, symmetry etc. for heavy atom location using SHELXD) and xx_fa.hkl (h,k,l,FA,sig(FA),alpha for both SHELXD and SHELXE). The starting phases for density modification are estimated as (heavy atom phase + alpha) in the simplified approach used by SHELXE, alpha is calculated by SHELXC from the anomalous and dispersive differences. For SAD alpha is 90º (I+ > I–) or 270º (I+ < I–), for SIR and RIP alpha is 0º or 180º and for SIRAS or MAD alpha may be anywhere in the range 0º to 360º.  

<p>The above command line could be used under UNIX or Windows; under UNIX the commands to run SHELXC, SHELXD and SHELXE and the instructions for SHELXC may also be combined into a single script file as shown in the following examples. In these scripts, the instructions start on the line after '<<EOF' and are terminated by 'EOF'. The instructions may be given in any order; CELL (unit-cell), SPAG (space group in PDB notation, spaces are ignored) and FIND (followed by the number of heavy atoms) must be given; the optional instructions SFAC, MIND, NTRY, SHEL, ESEL and DSUL, if present, are copied to the SHELXD input file. <br><br>

== MAD example ==

If the MAD experiment fails, one should insert the line 'SMAD' somewhere in the SHELXC input instructions and run the job again. This makes a MAD experiment into a SAD experiment in which a suitably weighted mean of the anomalous differences is employed and the dispersive differences are ignored. If the CC values in SHELXD come out better, this SAD approach is likely to give a better solution, but it may be then worth trying commenting out one or more of the PEAK, INFL, HREM and LREM commands to see if there is a further improvement (if just one remains, it should be renamed SAD).<br><br>

== SAD Example ==

The anomalous differences are extracted from the native data so only one data file is required. The sites specified by FIND consist of one methionine and 8 super-sulfurs, which are then resolved into disulfides using the DSUL instruction that is passed on to SHELXD (Debreczeni et al., Acta Cryst. D59 (2003) 2125-2132). Alternatively one could try to find the individual sulfurs with:<br>

Here the resolution cutoff has been reduced from 2.1 Å (which SHELXC would have suggested) to 2.0 Å to improve the chances of resolving the sulfurs. The SHEL, FIND, MIND and NTRY instructions are transferred to the file thau_fa.ins for the sulfur atom location with SHELXD. Note that the phases can be improved further in this case by using more SHELXE cycles than the usual 20. <br><br>

This involves the solution of the thaumatin structure using the above 1.55 Å data as native and 2.0 Å CuKalpha data from a quick iodide soak. SIRAS usually gives the best results for iodide soaks, but it is also possible in this case to use SIR (change ‘SIRA’ to ‘SIR’) or iodine SAD (change ‘SIRA’ to ‘SAD’). <br>

SHELXE has following modes of action (xx and yy are filename stems):<br>

xx.hkl contains native data, yy.hkl contains FA and alpha and should have been created using SHELXC or XPREP. xx.phi has .phs format (h,k,l,F,fom,phi in free format) and can be made by renaming a .phs output file from SHELXE, but only the starting phases are read from it; if a .phi file is read, the cell and symmetry are read from xx.ins and the native F-values are read from xx.hkl. xx.fcf (from a SHELXL structure refinement) provides cell, symmetry and starting phases. The output phases are written to xx.phs, the log file is written to xx.lst and, if -b is set, improved substructure phases are output to xx.pha and revised heavy atoms to xx.hat.<br>

SHELXE normally requires a few command line switches, e.g.<br>

would do 20 cycles density modification with a solvent content of 0.45, phasing from the first 8 heavy atoms in the yy.res file from SHELXD assuming that they are also present in the native structure (-h8), and then use the modified density to generate improved heavy atoms (-b). The switch -i may be added to invert the substructure (and if necessary the space group), this writes xx_i.phs instead of xx.phs etc., and so may be run in parallel. <br>