The heavy-atom sites are usually found using an automated method (see previous tutorial). It is important to remember that there is a hand ambiguity in the location of the sites. The search procedures are based purely on amplitude information. However, a given heavy-atom configuration and its inverse image give rise to identical diffraction amplitudes. The hand ambiguity cannot be resolved unless anomalous diffraction information is available. In general therefore the hand ambiguity can only be resolved by visual analysis of the resulting electron density map. The handedness of secondary structure elements such as alpha-helices or the chirality of amino acids (given sufficient resolution of the data) will indicate if the heavy atom sites need to be inverted.
In this example SIR phasing is performed with the CNS task file ir_phase_kuof.inp. We will perform SIR phasing using the native amplitudes and a uranyl derivative dataset. Three heavy atom sites have already been located using automated methods (see previous tutorial).
cns_solve < ir_phase_kuof.inp > ir_phase_kuof.out [3 minutes]
There are several output files:
ir_phase_kuof.summary
ir_phase_kuof.sdb
ir_phase_kuof.hkl
ir_phase_grad_kuof.hkl
The *.summary file contains information about the progress of
the SIR phasing procedure. The refined heavy-atom parameters are
stored in the *.sdb site database file. The refined phases
are written to the *.hkl files as Hendrickson-Lattman
coefficients, and also as centroid phases and corresponding figures of
merit. The Fourier coefficients for the gradient maps are stored in
the *_grad.hkl file. The use of the hkl file for
the detection of additional heavy-atom sites is shown in the following
tutorials.