THE MULTIWAVELENGTH ANOMALOUS SOLVENT CONTRAST (MASC) METHOD IN MACROMOLECULAR CRYSTALLOGRAPHY

The wavelength dependence of anomalous scattering of X-rays, due to atoms randomly dispersed in the solvent phase of a macromolecular crystal, is a way of producing solvent-density contrast variation with perfect isomorphism. The largest contrast variations are obtained by tuning the X-ray wavelength near an absorption edge of the anomalous-scattering species. In this method, which we call MASC, the anomalous partial structure is an extended uniform electron density, in contrast to the few punctual ordered scatterers in the multiwavelength anomalous-dispersion (MAD) method. MASC is, in principle, applicable to the determination of the molecular envelope and of low-resolution structure-factor phases. Structure factors F-lambda(+/-h) of an anomalous pair of reflections are expressed as a sum of two terms independent and dependent on wavelength, respectively. Squaring F-lambda(+/-h) leads to a set of equations which can be solved to give \G(h)\ and \F-O(h)\, the modulus of the envelope and of the total 'normal' structure factors, respectively, and Delta phi = (phi(F)- phi(G)). The moduli {IGI} behave like structure-factor amplitudes from small-molecule crystals, and the estimation of their phases can be carried out by statistical direct methods. Then, the phase of F-O(h) and finally the conventional (e.g. in vacuum) protein structure factor F-p(h) can be determined. As in the MAD method, the strength of MASC signals can be quantified by Bijvoet and dispersive ratios, for which practical expressions are derived in the case of zero contrast. The behaviour of these ratios at increasing resolution is discussed, using approximations for [Ic(h)t] and [I Delta(h)I], respectively, derived from Pored's law and assuming a random distribution of atoms in the solvent excluding volume. Expected values of anomalous ratios are calculated for a hypothetical MASC experiment based on the known three-dimensional structure of kallikrein A, using a solvent with 3.5 M ammonium selenate to ensure zero contrast, and wavelength tuning near the Se K-absorption edge. The main steps of a MASC experiment are discussed in the context of a MAD-like data collection optimized for accurate measurements of intensities of anomalous pairs at low resolution. Finally, the results of preliminary experiments on two protein crystals are reported. The first, a partial single-wavelength data collection, used anomalous scattering of selenium at the K edge and gave anomalous ratios with the expected behaviour. The second one, at three wavelengths, used anomalous scattering of ytterbium at the L(m) edge. In this case, effects from solvent as well as from ordered lanthanide ions were demonstrated.