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SHELX C/D/E: Difference between revisions
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== SHELXD == | == SHELXD == | ||
In general the critical parameters for locating heavy atoms with SHELXD are: | In general the critical parameters for locating heavy atoms with SHELXD are: | ||
=== Resolution cutoff (SHEL) === | |||
In the MAD case this is best determined by finding where the correlation coefficient between the signed anomalous differences for wavelengths with the highest anomalous signal (PEAK and HREM or PEAK and INFL) falls below about 30%. For SAD a less reliable guide is where the mean value of |ΔF|/σ(ΔF) falls below about 1.2 (a value of 0.8 would indicate pure noise), and for S-SAD with CuKα the data can be truncated where I/σ for the native data falls below 30. If unmerged data are used, SHELXC calculates a correlation coefficient between two randomly selected subsets of the signed anomalous differences; this is a better indicator because it does not require that the intensity esds are on an absolute scale, but it does require a reasonable redundancy and again the data can be truncated where it drops to below 30% (XDS and the CCP4 programs aimless/SCALA print a similar statistic). | |||
=== Number of sites (FIND) === | |||
The estimated number of sites (FIND) should be within about 20% of the true number. For SeMet or S-SAD phasing there should be a sharp drop in the occupancy after the last true site. For iodide soaks, a good rule of thumb is to start with a number of iodide sites equal to the number of amino-acids in the asymmetric unit divided by 15. If after SHELXD occupancy refinement the occupancy of the last site is more than 0.2 it might be worth increasing this number, and vice versa. | |||
It should be noted that the number of sites that SHELXD will search for is 40% higher than what is asked for by the user, in FIND. The reason for this is that there are often additional minor sites arising from heavy atoms, like Cl or Ca. So if you don't adjust FIND downwards, after an initial SHELXD run, such that the Nth site in the .res file has occupancy > 0.2, then you could either edit the .res file and remove the sites with occupancy < 0.2, or run SHELXE with -hN where N is the site number which has occupancy > 0.2 . | |||
=== Disulfides (DSUL) === | |||
If the resolution d (second parameter on SHEL card) is > 2.0Å the disulfide bonds may not fully resolved, but in the range 2.8>d>2.0 the DSUL instruction may be used to fit S−S units to the density. This can dramatically improve the final phase quality. If DSUL is used, the first MIND parameter should be set to -3.5 (so that each disulfide is found once only) and disulfides should be counted as single (super-sulfur) atoms for FIND (i.e. each disulfide given in DSUL counts as two atoms for FIND). | |||
=== Minimum distance between atoms (MIND) === | |||
A common 'user error' is to set MIND -3.5 even though the distances between heavy atoms are less than 3.5 Å. For example, in a Fe<sub>4</sub>S<sub>4</sub> cluster the Fe...Fe distance is about 2.7 Å, so MIND -2 would be appropriate. A disulfide bond has a length of 2.03 Å so then MIND -1.5 could be used to resolve the sulfur atoms, however if DSUL is used for this purpose MIND -3.5 is required. | |||
If heavy atoms can lie on special positions (as is the case with an iodide soak in a space group with twofold axes) the rejection of atoms on special positions should be switched off by giving the second MIND parameter as -0.1 (as in the above thaumatin example). | |||
=== Cubic space groups (PATS ''vs'' WEED) === | |||
In cubic space groups the Patterson seeding (PATS) is slow and less effective, it is recommended that 'PATS' is replaced by 'WEED 0.3'. | |||
=== Interpretation of results === | |||
For MAD, a CC of 40 to 50% indicates a good solution, for SAD etc. values around 30% may well be correct, especially if the same solution or group of solutions has the highest values of CC, CC(Weak) and PATFOM, and they are well separated from the values for the non-solutions. The CC values tend to increase as the resolution is lowered. Heavy atom soaks truncated to low resolution often give spuriously high CC values, but these 'solutions' can be recognized as false by their low CC(weak) values.<br> | For MAD, a CC of 40 to 50% indicates a good solution, for SAD etc. values around 30% may well be correct, especially if the same solution or group of solutions has the highest values of CC, CC(Weak) and PATFOM, and they are well separated from the values for the non-solutions. The CC values tend to increase as the resolution is lowered. Heavy atom soaks truncated to low resolution often give spuriously high CC values, but these 'solutions' can be recognized as false by their low CC(weak) values.<br> | ||