SAD phasing guidelines

Preparation of diffraction data

• Use all data.
• If possible, use the Wilson statistics to put the SAD data set on an absolute scale. This is important because phase refinement sometimes fails if the scale factor is very different from one. If Wilson statistics cannot be used because the high resolution limit of the SAD data is not at least 3 Å, a high resolution native data set should be used if available. Use the high resolution data set for the determination of the absolute scale factor, and scale the SAD data sets relative to this one.

Validation of diffraction data

• Use analyse_anom_diff_*.distr to check the anomalous signals by resolution shell and overall.
• Visual inspection of the Harker sections of anomalous difference Patterson maps is an alternative way to check anomalous signals. The Harker sections should show a number of outstanding peaks (depending on the number of anomalous scatterers).

Heavy atom search

• Search for only ~2/3 of the total number of expected sites.
• Resolution range: 15 Å and 4 to 3 Å.
• Use heavy_search.list to identify heavy atom configurations with outstanding correlation coefficients. Trials with a correlation coefficients greater than 1.5 sigma of all trials are flagged with a plus sign.

Heavy atom model refinement and completion

• Use all data unless there are serious doubts about the quality of the high resolution data.
• Use SAD data set as "Reference wavelength".
• Approximate values for f' and f" are usually sufficient.
• Use the anomalous difference Fourier map in combination with the (more powerful) gradient map to search for additional heavy atom sites.

Phase refinement, resolution of hand ambiguity, resolution of phase ambiguity

• Two heavy atom/phase refinements followed by density modification are carried out in parallel, one for each hand of the heavy atom configuration.
• Density modification is used to resolve the phase ambiguity.
• Visual inspection of the two density modified maps is used to resolve the hand ambiguity. One map should show significantly better solvent contrast and longer stretches of connected density.
• The higher the solvent content the more powerful is the density modification procedure.
• If visual inspection of the maps does not give a clear indication, review the sad_phase*.summary and density_modify*.list files for the phasing and density modification procedures, respectively. The phasing power in the low resolution shells should be greater than about two. The higher the better. The phasing power normally goes down in the high resolution shells. It is often not a good sign if the phasing power is more or less constant for all resolution shells, even if it is around two. For SAD, the phasing figure of merit tends to be relatively low even for good cases. A figure of merit of 0.3 in the low resolution shell could be a good result. The density modification figure of merit should be significantly different for the two hands of the heavy atom configuration. The higher density modification figure of merit should correspond to the correct map.