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Generalized Molecular Replacement

The following sections show how the 26-10 Fab fragment complexed with digoxin was solved by generalized molecular replacement (Brünger 1991c). The space group of the 26-10 Fab/digoxin crystals is (Strong 1990), with the b-axis unique, and a non-crystallographic twofold symmetry is present. The following strategy was employed to solve the structure:

  1. Self-rotation function.
  2. Modification of the elbow angle of a known Fab structure.
  3. Cross-rotation function with the modified Fab structure.
  4. Filtering the rotation function by PC-refinement.
  5. Analysis of the PC-refinement.
  6. Translation function for molecule A, using the PC-refined model.
  7. Translation function for molecule B.
  8. Combined translation function to determine the relative position between A,B.
  9. Rigid-body refinement.
In general, one has to try several different starting elbow angles (spaced approximately 10 apart) and repeat steps 3--6 until a good solution is found. This strategy is also applicable to other multidomain proteins or PC-refinements of other degrees of freedom. The modification of the Fab example input files should be straightforward. An overview of the strategy is shown in Fig. 17.1.

 
Figure 17.1: Overview of molecular replacement.  





Sat Mar 11 09:37:37 PST 1995