- Unfortunately the diffraction pattern alone is insufficient to determine the crystal
structure. Each diffraction maximum has both an amplitude and a phase associated
with it, and both need to be determined. But the phases are not directly measurable
in a diffraction experiment and must be estimated from further experiments.
This is usually done by the method of isomorphous replacement (MIR). The MIR
method requires at least two further crystals of the protein (derivatives), each being
crystallised in the presence of a different heavy-metal ion (e.g. Hg^2 þ,Cu^2 þ,Mn^2 þ).
Comparison of the diffraction patterns from the crystalline protein and the crystalline
heavy-metal atom derivative allows phases to be estimated. A more recent approach to
producing a heavy-metal derivative is to clone the protein of interest into a methionine
Fig. 8.4X-ray diffraction frame of data from a crystal of herpes simplex virus type 1 thymidine kinase,
complexed with substrate deoxythymidine, at 2 A ̊resolution. (Picture provided by John N. Champness,
Matthew S. Bennett and Mark R. Sanderson of King’s College London.)338 Protein structure, purification, characterisation and function analysis