Continuous density is seen for residues 249 to 253.
The free R-value of the model has now dropped to 30.6%. Inspection of the electron density map followed by manual rebuilding is now performed to improve the model. A cross-validated, sigma-A weighted, phase combined 2Fo-Fc map is calculated. Inspection of this map will help locate areas of the model which are in error. It would also be helpful to look at a difference map (at both positive and negative contour) at the same time. Here the map is calculated using the CNS task file model_map_2fofc.inp:
cns_solve < model_map_2fofc.inp > model_map_2fofc.out [2 minutes]
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 current
model and map.
The map is in general very good - the density clearly matches the model. However, there are regions which were not built initially which show traceable density:
|
| Sigma-A weighted, phase combined 2Fo-Fc map (at 1.5 sigma). Continuous density is seen for residues 249 to 253. |
The model is manually rebuilt in this region to add the missing residues 214 to 216 and 249 to 253 resulting in a new set of coordinates. These coordinates are then used to make new CNS coordinate and molecular topology files for further refinement. Here the CNS task file generate_easy_reb.inp is used:
cns_solve < generate_easy_reb.inp > generate_easy_reb.out [8 seconds]
The refinement is started with simulated annealing, which will correct errors introduced by the manual rebuilding and optimize the fit of the retraced region. As before a start temperature of 5000K is used. Here the CNS task file anneal_reb.inp is used:
cns_solve < anneal_reb.inp > anneal_reb.out [100 minutes]
At this point the free R-value is just below 32% (and the R-value is just below 29%). The model still might have some misplaced atoms (side chain atoms or small loops) but inspection of the maps suggest that the majority of the structure is correct. We therefore proceed with restrained individual B-factor refinement in preparation for automated water picking (see next section). The B-factor refinement is carried out with the CNS task file bindividual.inp:
cns_solve < bindividual.inp > bindividual.out [4 minutes]