Crystallography & NMR System

SAD Phasing - Density Modification - Selection of Map

Given the completed heavy-atom model, a new set of SAD phases can be computed. However, there are still two kinds of ambiguities which need to be resolved:

The hand or enantiomorph ambiguity of the heavy atom configuration.
This ambiguity is resolved by independent heavy atom refinement and SAD phasing with both choices.
The phase ambiguity.
This ambiguity is intrinsic to the SAD method. The phase probablity distribution for each reflection is bimodal. This means, there are two maxima in each probablity distribution. Density modification is used to resolve this ambiguity.

For the two independent SAD 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 SAD phasing jobs are identical except for the definitions of the file names for the heavy atom sites and the output file names.

      cns_solve < sad_phase2.inp > sad_phase2.out            [14 minutes]
      cns_solve < sad_phase2_flip.inp > sad_phase2_flip.out  [14 minutes]

For the density modification, it is necessary to have a good estimate of the solvent content. In a preliminary step, the solvent content is estimated by using matthews_coef.inp.

      cns_solve < matthews_coef.inp > matthews_coef.out  [< 1 second]

The estimated solvent content shown in matthews_coef.list is between 0.53 and 0.55. density_modify.inp and density_modify_flip.inp are modified accordingly by setting the solvent content parameter to 0.54.

      cns_solve < density_modify.inp > density_modify.out            [27 minutes]
      cns_solve < density_modify_flip.inp > density_modify_flip.out  [28 minutes]

The resulting output files are:

      density_modify*.list - listing file with statistics for
                             each modification cycle
      density_modify*.hkl  - reflection file with new Hendrickson-Lattman
                             coefficients
      density_modify*.map  - map file with modified electron density
      density_modify*.mask - file with definition of solvent mask

Electron density maps before solvent flipping are computed with modified copies of the fourier_map.inp task file.

      cns_solve < fourier_map_sad2.inp > fourier_map_sad2.out            [21 seconds]
      cns_solve < fourier_map_sad2_flip.inp > fourier_map_sad2_flip.out  [21 seconds]

If you have mapman installed, you can use the command

      map_to_omap *.map

to convert the CNS maps to a format which can be read into O. In O, enter @omac to read in the four maps. Portions of these maps are shown below.

**SAD phases**
Original sites	Flipped sites

**After solvent flipping**
Original sites	Flipped sites

Without density modification, it is practically impossible to decide which hand of the heavy-atom configuration is the correct one. However, after density modification the map based on the flipped sites shows long stretches of well connected density. In contrast, the map based on the original sites is uninterpretable.

Script to run this tutorial

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