Section 19.4.3.4. The triple isotopic substitution method

An innovation in the study of subunits in a reconstituted complex was introduced by Serdyuk et al. (1994), who devised a difference method to isolate the scattering from a single subunit. The method requires three particles with different deuteration levels in the subunit: one in which the subunit is heavily deuterated (contrast with the complex = ρ), one in which the subunit is not deuterated (0), and one in which the subunit is deuterated at an intermediate level (): where C is the scattering amplitude of the complex, S is the amplitude of the subunit, and F[CS] is the Fourier transform of the correlation function between the complex and the subunit. Scattering is measured (a) from an equimolar mixture of complexes with heavily deuterated and non-deuterated subunits and (b) from a sample of complexes with subunits with the intermediate level of deuteration. Subtraction of (b) from (a), weighted so that the two curves are equimolar, gives a net curve for the subunit alone (at half the scattering power that would be seen for a solution of the isolated subunits at the same concentration): The difference curve is not influenced by solvent composition, underlying order, concentration or interparticle interference effects. Thus, at the cost of some difficult biochemistry, the small-angle scattering of a subunit belonging to a large assembly can be observed in situ. In practice, the mixture is not equimolar, but is adjusted depending on the intermediate level of deuteration, relaxing some of the difficulty of the biochemistry.