The heavy-atom sites are usually found using an automated method (see previous tutorial). It is important to remember that there is an enantiomorph ambiguity in the location of the sites. The search procedures are based purely on amplitude information. However, a given heavy-atom configuration and its inverse image give rise to identical diffraction amplitudes. The enantiomorph ambiguity can be resolved by MAD phasing with both choices, followed by inspection of the resulting electron density maps. Only the correct hand will produce an interpretable map.
For the two independent MAD phasing jobs, two site database files are needed, one with the original heavy atom configuration and another with its inverse image. The file with the inverse image is generated with the flip_sites.inp task file.
cns_solve < flip_sites.inp > flip_sites.out [< 1 second]
The task files for the two independent MAD phasing jobs are identical
except for the definitions of the file names for the heavy atom sites
and the output file names.
cns_solve < mad_phase.inp > mad_phase.out [46 minutes]
cns_solve < mad_phase_flip.inp > mad_phase_flip.out [46 minutes]
There are two sets of output files, one for each input file:
mad_phase.summary mad_phase_flip.summary
mad_phase.fp mad_phase_flip.fp
mad_phase.sdb mad_phase_flip.sdb
mad_phase.hkl mad_phase_flip.hkl
mad_phase_grad.hkl mad_phase_flip_grad.hkl
The *.summary files contain information about the progress of
the MAD phasing procedure. The refined f' and f" can be found in
the *.fp files, and the refined heavy-atom parameters are
stored in the *.sdb site database files. The refined phases
are written to the *.hkl files as Hendrickson-Lattman
coefficients, and also as centroid phases and corresponding figures of
merit. The Fourier coefficients for the gradient maps are stored in the
*_flip_grad.hkl files. The use of the hkl files for
the determination of the correct hand and the detection of additional
heavy-atom sites is shown in the following tutorials.