Space group assignment in the presence of pseudosymmetry and twinning

The crystal structure of Ferrochelatase-1 (HemH) from Bacillus anthracis presented a moderately difficult case for space group assignment. The lattice parameters, merging statistics and systematic absences favoured the space group P2₁2₁2. A molecular replacement solution with a high contrast was found using the PDB entry 1ak1 as a search model (sequence identity of 73%). However, it was not possible to refine this solution (the lowest value of R-free was about 40%). The structure has been eventually solved, but in a monoclinic space group. In this tutorial we follow the whole procedure including revision of space group assignment.

The file p222.mtz contains unmerged HemH data presented in the space group P222. As these are unmerged data, the space group assignment only affects the order and indexing of observations and can be altered with no loss of information using pointless.

The file 1ak1.pdb was downloaded from the PDB and represents a crystal structure of Ferrochelatase-1 from Bacillus subtilis containing one protein molecule in the asymmetric unit. This structure will be used as a search model for molecular replacement.

1. Run pointless and scala to identify resolution cut-off and bad images
- Use the plot "R-merge vs. batch" for identifying bad images
- Use the plot "R-meas vs. 1/resol^2" to identify resolution cut-off

2. Rerun pointless and scala with bad data removed
- Use controls in the folder "Exclude data" in pointless
- Check the space group assigned in the default pointless run

3. Try to solve the structure using molecular replacement, model 1ak1, and merged data from (2)

4. Refine the structure (3) and take note of R and R-free, examine the refined model vs. electron density maps. Is there any potential for model improvement?

5. Run pointless selecting the second hit
- Check "choose previous solution" check-box.
- In the folder "Choose solution": Select "Choose solution from search by [Laue group solution number]" and type 2 in the corresponding text box.
- check the space group assigned in this pointless run

6. Run scala and check if systematic absences along b are convincing enough

7. Solve the structure using molecular replacement, model 1ak1, and merged data from (6)

8. Refine structure from (7), examine model vs. map, compare refinement statistics with one from step (4)