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SHELX C/D/E: Difference between revisions
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The -b switch in SHELXE causes updated heavy atom positions to be written to the file name.hat (or name_i.hat). This file can be copied or renamed to the .res file (which should be saved first!) and used to recycle the heavy atoms. The graphics program [[Coot]] should be able to deduce the space group name from the symmetry operators in this file, and so a very convenient way to obtain a map after running SHELXE is to start [[Coot]], read in 'coordinates' from the .hat or _i.hat file, and then input the phases from the .phs or _i.phs files and the phases of the heavy atom substructure from the .pha or _i.pha files. It is normally necessary to increase the sigma level of the latter map (by hitting '+' several times). This procedure even works correctly when the space group has been inverted by SHELXE! <br> | The -b switch in SHELXE causes updated heavy atom positions to be written to the file name.hat (or name_i.hat). This file can be copied or renamed to the .res file (which should be saved first!) and used to recycle the heavy atoms. The graphics program [[Coot]] should be able to deduce the space group name from the symmetry operators in this file, and so a very convenient way to obtain a map after running SHELXE is to start [[Coot]], read in 'coordinates' from the .hat or _i.hat file, and then input the phases from the .phs or _i.phs files and the phases of the heavy atom substructure from the .pha or _i.pha files. It is normally necessary to increase the sigma level of the latter map (by hitting '+' several times). This procedure even works correctly when the space group has been inverted by SHELXE! <br> | ||
Good quality MAD data, a high solvent content and/or high resolution for the native data can lead to maps of high quality that can be autotraced (e.g. with wARP) immediately. The .phs files contain h, k, l, F, fom, φ and σ(F) in free format and can be read directly into [[Coot]] or converted to CCP4 .mtz format using f2mtz, e.g. for further density modification exploiting NCS using the CCP4 program [[Pirate]]. Note that if the inverted heavy atom enantiomorph is the correct one, the corresponding phases are in the *_i.phs file and SHELXE may have inverted the space group (e.g. P4<sub>1</sub> to P4<sub>3</sub>), which should be taken into account when moving to other programs!<br> | Good quality MAD data, a high solvent content and/or high resolution for the native data can lead to maps of high quality that can be autotraced (e.g. with wARP) immediately. The .phs files contain h, k, l, F, fom, φ and σ(F) in free format and can be read directly into [[Coot]] or converted to CCP4 .mtz format using [[f2mtz]], e.g. for further density modification exploiting NCS using the CCP4 program [[Pirate]]. Note that if the inverted heavy atom enantiomorph is the correct one, the corresponding phases are in the *_i.phs file and SHELXE may have inverted the space group (e.g. P4<sub>1</sub> to P4<sub>3</sub>), which should be taken into account when moving to other programs!<br> | ||
=== The free lunch algorithm (FLA) === | === The free lunch algorithm (FLA) === | ||
The new switch -e may be used to extrapolate the data to the specified resolution (the 'free lunch algorithm', based closely on work by the Bari group | The new switch -e may be used to extrapolate the data to the specified resolution (the '''''free lunch algorithm'''''), based closely on work by the Bari group (Caliandro ''et al''., ''Acta Crystallogr''. (2005) '''D61''', 556-565) and independently implemented in the program [[Acorn]] (Yao ''et al''., (2005) ''Acta Crystallogr''. '''D61''', 1465-1475): -e1.0 can produce spectacular results when applied to data collected to 1.6 to 2.0 Å, but since a large number of cycles is required (-m400) and the 'contrast' and 'connectivity' become unreliable (the pseudo-free CC is the only reliable map quality indicator when the FLA is used), it may be best to establish the substructure enantiomorph and solvent content without -e first. The default setting when -e is not specified is to fill in missing low and medium resolution data but not to extrapolate to higher resolution than actually measured (to switch off this filling in, use -e999). The resolution requirements for the FLA still need to be explored, but so far there have been no reports of it causing a deterioration in map quality, and in a few cases the mean phase error was reduced by as much as 30º relative to density modification without it.<br> | ||
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shelxe jia jia_fa -s0.6 -m20 -i | 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 | 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 F<sub>A</sub>-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> | 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> | ||
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=== SAD === | === SAD === | ||
This example of thaumatin phasing by means of the native sulfur anomalous signal (Debreczeni et al., Acta | This example of thaumatin phasing by means of the native sulfur anomalous signal (Debreczeni et al., ''Acta Crystallogr''. '''D59''' (2003) 688-696) uses 1.55 Å in-house CuKα data: | ||
shelxc thau <<EOF | shelxc thau <<EOF | ||
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shelxe thau thau_fa -h -s0.5 -m20 –i | 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 | 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 Crystallogr''. '''D59''' (2003) 2125-2132). Alternatively one could try to find the individual sulfurs with:<br> | ||
SHEL 999 2.0 | SHEL 999 2.0 | ||
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=== SIRAS === | === SIRAS === | ||
This involves the solution of the thaumatin structure using the above 1.55 Å data as native and 2.0 Å | This involves the solution of the thaumatin structure using the above 1.55 Å data as native and 2.0 Å CuKα 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 | shelxc thaui <<EOF | ||