3. Calculating Phases
When solving for heavy atom sites it is important to double-check them with cross-difference fouriers to verify that the origins are correlated. For now, I'll ask you to accept on faith that this is true for the two heavy atom solutions I will give you (below). Now all we have to do is to plug in the fractional coordinates for each heavy atom in each derivative (in our case their is only one site per derivative) and then refine these sites, ultimately with the aim of calculating multiple isomorphous replacement phases supplemented by their anomalous scattering contributions.
To calculate phases, we will use a maximum likelihood method implemented in the CCP4 program mlphare. The input file contains entries for the x, y and z fractional coordinates for each heavy atom, its estimated real and imaginary occupancies (the latter is an empirically-adjustable measure of its anomalous scattering power) and a temperature factor (loosely this is correlated with the disorder of the atom but in reality it is hard to read in too much physical significance at this stage). The real occupancy of a fully-occupied site will be 1.0 iff the data are on an absolute scale. They are not, however, so again these numbers are relative. The anomalous occupancy will approach a factor of three times the real occupancy during the refinement. This indicates that the anomalous scattering is contributing to the phase estimates. Here is what I have as input for the two heavy atom sites:
123: 0.405 0.098 0.305 0.200 0.500 BFAC 50.000
124: 0.417 0.196 0.238 0.200 0.500 BFAC 50.000
You can (and should) check those coordinates to verify that they correspond to their respective Patterson maps.
To make your life simple, I've given you an mlphare input file for the space group P3121, which is the correct space group. In reality, we have a 50% chance of being wrong, because P3221, its enantiomorph (mirror image) is equally probable a priori. Anomalous scattering data from MORE THAN ONE derivative enables one to distinguish between the two space groups. The anomalous occupancies in P3221 will be considerably smaller. Try it, but don't forget to change the handedness of the heavy-atom positions if you do!
mlphare is a CCP4 program that calculates phases based upon a maximum likelihood phase calculation algorithm.
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