Multiple isomorphous replacement


Multiple isomorphous replacement is historically the most common approach to solving the phase problem in X-ray crystallography studies of proteins. For protein crystals this method is conducted by soaking the crystal of a sample to be analyzed with a heavy atom solution or co-crystallization with the heavy atom. The addition of the heavy atom to the structure should not affect the crystal formation or unit cell dimensions in comparison to its native form, hence, they should be isomorphic.
Data sets from the native and heavy-atom derivative of the sample are first collected. Then the interpretation of the Patterson difference map reveals the heavy atom's location in the unit cell. This allows both the amplitude and the phase of the heavy-atom contribution to be determined. Since the structure factor of the heavy atom derivative of the crystal is the vector sum of the lone heavy atom and the native crystal then the phase of the native Fp and Fph vectors can be solved geometrically.
At least two isomorphous derivatives must be evaluated since using only one will give two possible phases.

Development

Single Isomorphous Replacement (SIR)

Early demonstrations of isomorphous replacement in crystallography come from Cork, John Monteath Robertson, and others. An early demonstration of isomorphous replacement in crystallography came in 1927 with a paper reporting the x-ray crystal structures of a series of alum compounds from James M. Cork. The alum compounds studied had the general formula A.B.2.12H2O, where A was a monovalent metallic ion, B was a trivalent metallic ion and S was usually sulfur, but could also be selenium or tellurium. Because the alum crystals were largely isomorphous when the heavy atoms were changed out, they could be phased by isomorphous replacement. Fourier analysis was used to find the heavy atom positions.
The first demonstation of isomorphous replacement in protein crystallography was in 1954 with a paper from David W. Green, Vernon Ingram, and Max Perutz.

Multiple Isomorphous Replacement (MIR)

Examples

Some examples of heavy atoms used in protein MIR:
Two methods for providing the needed phasing information by introducing heavy atoms into isomorphous crystals: