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== Overview ==
== SHELXC ==


'''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''' 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).
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<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>
<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 ===
== SHELXD ==


shelxc jia <<EOF
=== critical parameters ===
NAT jia_nat.hkl
HREM jia_hrem.sca
PEAK jia_peak.sca
INFL jia_infl.sca
LREM jia_lrem.sca
CELL 96.00 120.00 166.13 90 90 90
SPAG C2221
FIND 8
NTRY 10
EOF
shelxd jia_fa
shelxe jia jia_fa -s0.6 -m20
shelxe jia jia_fa -s0.6 -m20 -i
 
In this example (kindly donated by Zbigniew Dauter; Li et al., Nature Struct. Biol. 7 (2000) 555-559), Se-Met MAD data at four wavelengths are used to calculated the FA-values and phase shifts that are written to the file jia_fa.hkl. The native (S-Met) data are read from jia_nat.hkl and written to jia.hkl. The file jia_fa.ins is prepared using the given cell, space group, FIND and NTRY instructions as well as a suitable SHEL command to truncate the resolution. SHELXD then searches for 8 (FIND) selenium atoms using 10 attempts (NTRY), and SHELXE is run for 20 cycles (-m) of density modification for both heavy atom enantiomorphs (-i inverts) with a solvent content (-s) of 0.6. The protein phases are written to jia.phs and jia_i.phs resp.  If NAT is not specified, SHELXC would analyze the four MAD datasets to generate the (SeMet) native data jia.hkl, in which case -h should be specified for SHELXE since the selenium atoms are present in the ‘native’ structure. For MAD at least two wavelengths are required, at least one of which should be PEAK or INFL.
 
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 ===
 
This example of thaumatin phasing by means of the native sulfur anomalous signal (Debreczeni et al., Acta Cryst. D59 (2003) 688-696) uses 1.55 Å in-house CuKalpha data:
 
shelxc thau <<EOF
SAD thau-nat.hkl
CELL 58.036 58.036 151.29 90 90 90
SPAG P41212
FIND 9
DSUL 8
MIND –3.5
NTRY 100
EOF
shelxd thau_fa
shelxe thau thau_fa -h -s0.5 -m20
shelxe thau thau_fa -h -s0.5 -m20 –i
 
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>
 
SHEL 999 2.0
FIND 17
MIND –1.7
 
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>
 
=== SIRAS example ===
 
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>
 
shelxc thaui <<EOF
NAT thau-nat.hkl
SIRA thau-iod.hkl
CELL 58.036 58.036 151.29 90 90 90
SPAG P41212
FIND 17
NTRY 10
MIND –3.5 –0.1
EOF
shelxd thaui_fa
shelxe thaui thaui_fa -s0.5 –m20
shelxe thaui thaui_fa -s0.5 –m20 -i
 
 
== SHELXD : critical parameters ==


In general the critical parameters for locating heavy atoms with SHELXD are:
In general the critical parameters for locating heavy atoms with SHELXD are:
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RIP (without using anomalous scattering) or RIPAS (like SIRAS, assuming that the anomalous atoms are also those most sensitive to radiation damage) can be capable of solving difficult structures. A typical procedure on a third generation synchrotron beamline is to collect the 'before' dataset with an attenuator in the beam, then to fry the crystal for a couple of minutes with the unattenuated beam, and finally to collect an 'after' dataset with the attenuator in. In the SHELXC instructions, the 'before' data are called 'NAT' or 'BEFORE' and the 'after' data are called 'RIP' or 'AFTER'. The critical parameter is the scale factor applied to the 'after' data after both datasets have been brought onto a common scale. This is set by the SHELXC instruction 'DSCA' and should usually be in the range 0.95 to 1.00. This scale factor may also be used for SIR and SIRAS, where it is applied to the native data, but it appears to be less critical than for RIP. For RIPAS, the 'after' data should be called 'RIPA' and the 'RIPW' instruction specifies the weight w (default 0.6) for the anomalous contribution from the 'before' dataset (a weight 1–w is applied to the 'after' data).
RIP (without using anomalous scattering) or RIPAS (like SIRAS, assuming that the anomalous atoms are also those most sensitive to radiation damage) can be capable of solving difficult structures. A typical procedure on a third generation synchrotron beamline is to collect the 'before' dataset with an attenuator in the beam, then to fry the crystal for a couple of minutes with the unattenuated beam, and finally to collect an 'after' dataset with the attenuator in. In the SHELXC instructions, the 'before' data are called 'NAT' or 'BEFORE' and the 'after' data are called 'RIP' or 'AFTER'. The critical parameter is the scale factor applied to the 'after' data after both datasets have been brought onto a common scale. This is set by the SHELXC instruction 'DSCA' and should usually be in the range 0.95 to 1.00. This scale factor may also be used for SIR and SIRAS, where it is applied to the native data, but it appears to be less critical than for RIP. For RIPAS, the 'after' data should be called 'RIPA' and the 'RIPW' instruction specifies the weight w (default 0.6) for the anomalous contribution from the 'before' dataset (a weight 1–w is applied to the 'after' data).
In RIP or RIPAS phase determination is usually necessary to recycle the 'heavy atom' sites by renaming the output .hat (or _i.hat) file as .res and rerunning SHELXE. It is advisable to edit this file so as to retain the stronger negative sites, these may well correspond to the new positions of displaced atoms. SHELXE can read negative occupancies but SHELXD can only search for positive atoms. It should be noted that in a pure RIP experiment, both hands of the radiation damage substructure will give the same figures of merit, but one will lead to an electron density map that is a mirror image of the true map (the helices will go the wrong way round). <br>
In RIP or RIPAS phase determination is usually necessary to recycle the 'heavy atom' sites by renaming the output .hat (or _i.hat) file as .res and rerunning SHELXE. It is advisable to edit this file so as to retain the stronger negative sites, these may well correspond to the new positions of displaced atoms. SHELXE can read negative occupancies but SHELXD can only search for positive atoms. It should be noted that in a pure RIP experiment, both hands of the radiation damage substructure will give the same figures of merit, but one will lead to an electron density map that is a mirror image of the true map (the helices will go the wrong way round). <br>
== Examples ==
=== MAD ===
shelxc jia <<EOF
NAT jia_nat.hkl
HREM jia_hrem.sca
PEAK jia_peak.sca
INFL jia_infl.sca
LREM jia_lrem.sca
CELL 96.00 120.00 166.13 90 90 90
SPAG C2221
FIND 8
NTRY 10
EOF
shelxd jia_fa
shelxe jia jia_fa -s0.6 -m20
shelxe jia jia_fa -s0.6 -m20 -i
In this example (kindly donated by Zbigniew Dauter; Li et al., Nature Struct. Biol. 7 (2000) 555-559), Se-Met MAD data at four wavelengths are used to calculated the FA-values and phase shifts that are written to the file jia_fa.hkl. The native (S-Met) data are read from jia_nat.hkl and written to jia.hkl. The file jia_fa.ins is prepared using the given cell, space group, FIND and NTRY instructions as well as a suitable SHEL command to truncate the resolution. SHELXD then searches for 8 (FIND) selenium atoms using 10 attempts (NTRY), and SHELXE is run for 20 cycles (-m) of density modification for both heavy atom enantiomorphs (-i inverts) with a solvent content (-s) of 0.6. The protein phases are written to jia.phs and jia_i.phs resp.  If NAT is not specified, SHELXC would analyze the four MAD datasets to generate the (SeMet) native data jia.hkl, in which case -h should be specified for SHELXE since the selenium atoms are present in the ‘native’ structure. For MAD at least two wavelengths are required, at least one of which should be PEAK or INFL.
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 ===
This example of thaumatin phasing by means of the native sulfur anomalous signal (Debreczeni et al., Acta Cryst. D59 (2003) 688-696) uses 1.55 Å in-house CuKalpha data:
shelxc thau <<EOF
SAD thau-nat.hkl
CELL 58.036 58.036 151.29 90 90 90
SPAG P41212
FIND 9
DSUL 8
MIND –3.5
NTRY 100
EOF
shelxd thau_fa
shelxe thau thau_fa -h -s0.5 -m20
shelxe thau thau_fa -h -s0.5 -m20 –i
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>
SHEL 999 2.0
FIND 17
MIND –1.7
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>
=== SIRAS ===
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>
shelxc thaui <<EOF
NAT thau-nat.hkl
SIRA thau-iod.hkl
CELL 58.036 58.036 151.29 90 90 90
SPAG P41212
FIND 17
NTRY 10
MIND –3.5 –0.1
EOF
shelxd thaui_fa
shelxe thaui thaui_fa -s0.5 –m20
shelxe thaui thaui_fa -s0.5 –m20 -i




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