W0072

Neutron “Small-Angle” Crystallography. Contrast Variation in Single Crystals of Biological Macromolecules. Peter A. Timmins, Institut Laue-Langevin, BP156, 38042 Grenoble Cedex 9, France.

The contrast variation technique is best known through its application in neutron small-angle scattering. The concept was, however, first applied in the early days of X-ray protein crystallography when Bragg and Perutz determined the molecular envelope of haemoglobin by soaking crystals in sucrose in order to change the electron density and hence contrast of the solvent with respect to the protein.

By analogy contrast variation using H₂O/D₂O mixtures or molecule specific deuteration can be used to determine molecular envelopes in neutron crystallography. This is particularly useful when one component of a molecular complex is disordered in the crystal. Such is the case for example in many viruses where the protein coat may be perfectly ordered but due to symmetry mismatch the nucleic acid is disordered and invisible in standard high-resolution x-ray crystallographic studies. A similar effect is seen in crystals of membrane proteins where, although the protein itself is well ordered and its structure can be obtained at high resolution, the detergent used to solubilize the protein is fluid and disordered and hence invisible in X-ray maps.

In order to measure diffraction from small crystals of large unit cell proteins optimised instrumentation has been developed exploiting the ILL’s high flux of long-wavelength neutrons. The phase problem can be solved in a manner similar to single isomorphous replacement exploiting the linear relationship between phase and contrast if the structure of one component in the crystal is known.

The structure determination of detergent/membrane-protein complexes is a particularly good example of the low-resolution crystallographic method illustrating the unique power of neutrons in identifying molecular interactions in crystals. A number of examples in which protein-protein, detergent-detergent and protein-detergent interactions have varying significance will be shown.