Multiple Isomorphous Replacement

Phase information has to be obtained or deduced to solve crystal structures. This information can be obtained largely ab initio for small molecules that diffract to very high resolution. Proteins tend to diffract to moderate resolution, and the phase information has to be obtained independently. One method is Multiple Isomorphous Replacement.

Crystals of a protein sample are first grown without any specific additives, and diffraction recorded from them. Then, some crystals are soaked in a solution containing a dissolved 'heavy-atom' salt, or derivative, usually elements from the periodic table that are heavier than the C, N, O and S typically found in proteins. These heavier elements can bind to the protein in specific sites without causing any structural change. This gives diffraction intensities that are different from those of the additive-free crystal. These 'isomorphous' differences are then measured and used to obtain the positions of the heavy atoms in the crystal structure, which in turn gives a starting point for phase determination, leading to structure solution.

The heavy atom derivatives can be prepared by setting up a new crystallisation of the protein from solutions already containing the heavier element salt, co-crystallisation.

The information given by one derivative has a 'right-left' ambiguity, which can be broken by preparing another derivative. Pooling the information selects the correct solution. But often similar derivatives give the same information. So more than one or two derivatives are usually needed, besides the native form. Hence the use of 'multiple' in the name.

The isomorphous difference is larger in magnitude than the anomalous signal, but is not sufficient on its own. However, if the two differences can be measured in one experiment, then together with the native experiment, that would be sufficient to solve the structure.

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