7. Revising point group assignment

Project directory: 7_symm

Reflection data: p222.mtz

Structure file: 1ak1.pdb

7.1. Introduction

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 P 21 21 2. 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 in MTZ-file only defines the sort order and indexing of observations and can be altered with no loss of information using Pointless.

The space group assignment as well as truncation of bad data will be done using Aimless pipeline, which includes Pointless for space group assignment and corresponding sorting of reflections, Aimless for data scaling and merging, and CTruncate for analysis of merged data and conversion of intensities to structure amplitudes. The Aimless-pipeline task interface can be started from here: CCP4I > Data Reduction and Analysis > Symmetry, Scale and Merge (Aimless).

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.

7.2. Attempt at structure solution in an incorrect space group

1. Run Aimless pipeline to identify resolution cut-off and bad images

- Use the plot "R-merge vs. batch" for identifying bad sector(s) of images

- Use the plot "R-meas vs. 1/resol^2" to identify resolution cut-off (R-meas of about 0.5 in high resolution shell is acceptable)

2. Rerun Aimless pipeline indicating which parts of data should be excluded

- Use controls in the folder "Resolution and batch exclusions"

- Compare results with (1)

In addition, check if there are indications of incorrect space group assignment

- can a crystal with the assigned space group be twinned?

- do twinning tests suggest any twinning?

- are there any significant intensities along putative screw axes which should not be present?

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?

7.3. Structure solution

5. Run Aimless pipeline selecting the second hit from the list of point groups

· Press "Customise symmetry determination" check-button.

· Press "Choose a previous solution" check-button.

· In the folder "Options for Pointless": Select "Choose solution from search by [Laue group solution number]" and type 2 in the corresponding text box.

· Check what is the new space group

· Check if systematic absences along b* are convincing enough

6. Solve the structure using molecular replacement, model 1ak1, and merged data from (5)

8. Refine the structure from (6), examine model vs. map and compare the refinement statistics with the one from step (4)