Date Published: February 01, 2017
Publisher: International Union of Crystallography
Author(s): Dorothee Liebschner, Pavel V. Afonine, Nigel W. Moriarty, Billy K. Poon, Oleg V. Sobolev, Thomas C. Terwilliger, Paul D. Adams.
Residual OMIT maps can be improved by the selective exclusion of bulk solvent from the OMIT region.
OMIT maps (Bhat & Cohen, 1984 ▸) are a widely used tool to verify whether a certain region of a model in a crystallographic map has sufficient density to justify its presence in the model. An OMIT map is calculated by excluding the atoms in question from the model and is especially useful to verify the presence of ligands, solvent molecules, alternative conformations and residues with weak electron density. Various kinds of OMIT maps have been proposed (Bhat, 1988 ▸; Hodel et al., 1992 ▸; Vellieux & Dijkstra, 1997 ▸; Gunčar et al., 2000 ▸; Terwilliger, Grosse-Kunstleve, Afonine, Moriarty, Adams et al., 2008 ▸; Pražnikar et al., 2009 ▸). These maps have their advantages and disadvantages. In the case of ligands and alternative conformations, it is desirable to first build and correct the rest of the model before placing a ligand or building difficult-to-interpret residues (‘discovery map’; Tronrud, 2008 ▸).
The calculation of polder OMIT maps consists of several stages (Fig. 3 ▸). Firstly, the OMIT region of the unit-cell volume is identified by selecting a group of atoms in the input model that are located in the OMIT region. An intersection of spheres of radius 5 Å around each selected atom is used to mark this region. The choice of 5 Å for the sphere radius is rather arbitrary and is based on two requirements. One is that the OMIT region needs to be large enough to avoid biasing the map by the shape of the masking atoms. The other is that the OMIT region should not remove too much scattering from the model because otherwise it will be damaging to the map. This is especially important for weak features in the map as they may be particularly susceptible to model deterioration. The results from calculations testing different radii for bulk-solvent mask exclusion are presented in the Supporting Information.
In this section, several examples of the utility of polder maps are presented.
Several approaches have been proposed to decrease the influence of flat bulk solvent in OMIT maps: not using a bulk-solvent model at all and truncating the data at ∼5–6 Å resolution or employing a solvent model which does not use a mask, such as the Babinet model. Fig. 5 ▸ shows these maps for ligand MES 88 of PDB entry 1aba. For the map computed without using any bulk-solvent model, the resolution was truncated at 5 Å. This resolution cutoff was obtained by comparing the curves of the R factor versus resolution calculated using the flat bulk-solvent model and without using any bulk-solvent model (Fig. 8 ▸). The curves are similar up to 4 Å resolution and begin to diverge at about 5 Å.
If the omitted region is surrounded by other atomic features of the model, such as a ligand in a compact binding pocket, the residual density revealed by the polder map might show behavior similar to biased OMIT maps: such density may correspond to either bulk solvent or ordered atoms. The reason is that in tightly packed environments bulk solvent or ligands are likely to mimic the shape of the pocket.
It is often suspected that omitting atoms does not entirely remove model bias (Hodel et al., 1992 ▸). It is therefore common to carry out several rounds of refinement, optionally adding simulated annealing (SA) to remove the ‘memory’ of the atoms to be omitted (Rupp, 2009 ▸; Terwilliger, Grosse-Kunstleve, Afonine, Moriarty, Zwart et al., 2008 ▸; Brünger et al., 1998 ▸). To compare the result of the polder procedure with a standard SA map, SA refinement was performed with the simulated_annealing=True option for the first macrocycle in phenix.refine (Afonine et al., 2012 ▸) for model 4opi (without ligand GRG 502). The OMIT map and the polder map for ligand GRG 502 are displayed in Fig. 10 ▸. Similar to the results discussed in §3.1.1, the SA OMIT map (Fig. 10 ▸a) has much less clear ligand density than the SA polder map (Fig. 10 ▸b).
The flat bulk-solvent model affects OMIT maps. To avoid its influence, a new tool, phenix.polder, has been developed as part of the PHENIX software suite. The tool calculates OMIT maps by not only excluding the selected atoms but also preventing the bulk-solvent mask from penetrating the region in question. As shown by several examples, phenix.polder is useful in cases where the density of the selected atoms is weak and possibly obscured by the bulk solvent. phenix.polder produces less biased maps than procedures in which the atoms are simply removed from the model or where the atom-selection occupancy is set to zero and included in the solvent-mask calculation. In the latter case, the resulting difference density can have a similar shape as the selected atoms. In the polder procedure, a larger volume from the bulk solvent is excluded and therefore prevents the misinterpretation of bulk-solvent density as OMIT density, making it a map-improvement technique that is suitable for parts of the structure with weak density. The program is available as from the command line as well as in the PHENIX GUI.