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SHELX C/D/E: Difference between revisions
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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> | 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> | ||
<font face="Courier New"><b> | <font face="Courier New"><b> | ||
SHEL 999 2.0<br> | SHEL 999 2.0<br> | ||
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RIP (radiation damage induced phasing) can be regarded as a sort of isomorphous replacement where the 'after' dataset has lost a few atoms that are particularly susceptible to radiation damage. In fact, many structures have solved unintentionally with a helping hand from RIP! In a MAD experiment, provided that the 'inflection point' dataset is collected last from the same crystal, the radiation damage has the effect of making f' for the MAD element at this wavelength even more negative than usual, enhancing the dispersive part of the MAD signal. This is especially true of bromine MAD on bromouracil derivatives, because the radiation near the bromine absorption edge appears to be particularly effective at breaking the bromine-carbon bonds irreversibly. Of course if the inflection data are collected first the RIP and dispersive component of the MAD signal will tend to cancel one another, causing the MAD analysis to fail, although SAD may still be able to solve the structure (also a common scenario). | RIP (radiation damage induced phasing) can be regarded as a sort of isomorphous replacement where the 'after' dataset has lost a few atoms that are particularly susceptible to radiation damage. In fact, many structures have solved unintentionally with a helping hand from RIP! In a MAD experiment, provided that the 'inflection point' dataset is collected last from the same crystal, the radiation damage has the effect of making f' for the MAD element at this wavelength even more negative than usual, enhancing the dispersive part of the MAD signal. This is especially true of bromine MAD on bromouracil derivatives, because the radiation near the bromine absorption edge appears to be particularly effective at breaking the bromine-carbon bonds irreversibly. Of course if the inflection data are collected first the RIP and dispersive component of the MAD signal will tend to cancel one another, causing the MAD analysis to fail, although SAD may still be able to solve the structure (also a common scenario). | ||
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) | 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> | ||
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Sheldrick, G.M. (2002), "Macromolecular phasing with SHELXE", ''Z. Kristallogr''. '''217''', 644-650 [''The definitive reference for SHELXE, usually cited wrongly''].<br> | Sheldrick, G.M. (2002), "Macromolecular phasing with SHELXE", ''Z. Kristallogr''. '''217''', 644-650 [''The definitive reference for SHELXE, usually cited wrongly''].<br> | ||
Nanao, M.H., Sheldrick, G.M. & Ravelli, R.B.G. (2005). "Improving radiation-damage substructures for RIP", ''Acta Crystallogr''. '''D61''', 1227-1237 [''Practical details of RIP phasing with SHELXC/D/E''].<br> | |||
Uson, I., Stevenson, C.E.M., Lawson, D.M. & Sheldrick, G.M. (2007). "Structure determination of the O-methyltransferase NovP using the `free lunch algorithm' as implemented in SHELXE", ''Acta Crystallogr''. '''D63''', 1069-1074 [''Implementation of the FLA in SHELXE''].<br> | Uson, I., Stevenson, C.E.M., Lawson, D.M. & Sheldrick, G.M. (2007). "Structure determination of the O-methyltransferase NovP using the `free lunch algorithm' as implemented in SHELXE", ''Acta Crystallogr''. '''D63''', 1069-1074 [''Implementation of the FLA in SHELXE''].<br> | ||