Laboratory of Macromolecular Crystallography
ac_predicton and shift_plot: programs for analyzing atomic shifts in unrestrained refinement
Unrestrained refinement is stable for the vast majority of atoms when working at atomic resolution. Nevertheless geometrical restraints should be held in refinement for residues that are present in several (alternative) conformations in the crystal used for the X-ray experiment; in the other case such residues deteriorate significantly. We believe that on the contrary, large distortion of a residue in unrestrained refinement may hint the presence of alternative conformations for this residue. To get these hints in a routine way we suggest two methods to analyze shifts of atomic centers resulted from several cycles of unrestrained refinement. The first method is a simple diagram plotting the values of atomic shifts against the residue number. It may give an idea of the crystallographic order of different parts of the structure at qualitative level.
The second method is decision-making procedure which compose a list of residues that are likely to be present in alternative conformations or disordered and so should be checked thoroughly with Fourier syntheses, and included into the model with alternative conformations, when necessary. The parameters of the suggested procedures were estimated with the use of 203 PDB structures refined at resolution better than 1.2A.
Both programs are designed to analyze atomic shifts during unrestrained refinement. Therefore the user should perform such
a refinement and provide 2 pdb-files: the model before refinement and the model after refinement.
To analyze a particular model, we offer to apply the Trial Unrestrained
Refinement (TUR) consisted of 3 macro-cycles of unrestrained refinement
with phenix.refine or 10 cycles of unrestrained refinement with Refmac.
Shift_plot will produce a diagram of atomic shifts vs. residue number. The largest shifts indicate the residues that are considered in this approach as the main candidates to be present in alternative conformations, and should be checked first. Shift_plot also may be used to analyze atomic shifts resulted from any other model manipulation or refinement. It matches atoms with the same:
- atom serial number;
- atom name;
- residue name;
- chain ID;
- residue number;
- insertion code.
AC_prediction implements automatic decision-making procedure that classifies every residue as ‘single conformation’ or ‘alternative conformations’ (SC and AC below). The procedure is based on the observation that SC-residues and AC-residues have different mobility in unrestrained refinement. The decision is made either by comparing the observed atomic shifts with a predetermined threshold, or comparing the probabilities of such shifts for SC and AC-residues.
The use of different refinement programs
Different programs for crystallographic refinement have their own internal procedures for stabilizing the refinement that affect the values of obtained atomic shifts. Therefore, the values of the thresholds and the empirical probability distributions for different types of atoms are specific to a particular refinement program. At the same time, the tendency to have large values of shifts for the atoms occupying alternate positions takes place with other programs as well. As a result the diagrams of atomic shifts are more software-independent and can be used to analyze the output of any refinement program.
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