MAD Phasing Strategies Explored with a Brominated Oligonucleotide Crystal at 1.65Å Resolution

Abstract

The crystal structure of a brominated oligonucleotide d(CGCG(Br)CG), chemical formula C(114)N(48)O(68)P(10)Br(2), has been analysed by multiwavelength anomalous dispersion (MAD) methods. The oligonucleotide crystallizes in space group P2(1)2(1)2(1) with a = 17.97, b = 30.98, c = 44.85 A, alpha = beta = gamma 90 degrees . Data to a resolution of 1.65 A were collected at four wavelengths about the K-absorption edge of the bromine atom (lambda(1) = 0.9323 A, a reference wavelength at the long-wavelength side of the edge; lambda(2) = 0.9192 A, at the absorption-edge inflection point; lambda(3) = 0.9185 A, at the ;white line' absorption maximum; lambda(4) = 0.8983 A, a reference wavelength at the short-wavelength side) using synchrotron radiation at Station PX9.5, SRS, Daresbury. Multiwavelength data could be collected on a single-crystal as the sample was radiation stable. Anomalous and dispersive Patterson maps were readily interpretable to give the bromine anomalous scatterer positions. Phase calculations to 1.65 A, resolution, using all four wavelengths, gave a figure of merit of 0.825 for 2454 reflections. The electron-density map was readily interpretable showing excellent connectivity for the sugar/phosphate backbone and each base was easily characterized. The two nucleotide strands paired up as expected in an antiparallel Watson-Crick-type manner. The structure was refined to 1.65 A using all the data (R-factor = 17.0% based on 3151 reflections, with a data-to-parameter ratio of 2.6). In addition to the four-wavelength analysis, a variety of other phasing strategies, and the associated quality of the resulting electron-density maps, were compared. These included use of either of the reference wavelength data sets in the two possible three-wavelength phasing combinations to assess their relative effectiveness. Moreover, the time dependence upon measuring the Bijvoet differences and its effect upon phasing was also investigated. Finally, the use of only two wavelengths, including Friedel pairs, is demonstrated (the theoretical minimum case); this is of particular interest when considering overall beam time needs and is clearly a feasible experimental strategy, as shown here.

A MAD experiment performed at the white line of the iridiumLIIIabsorption edge in lysozyme

“…It is evident that even a well measured two-wavelength solution can still provide good phasing information as can be seen from solution (j). This supports the observations of Peterson et al (1996), who successfully obtained a fully interpretable electron-density map of a brominated oligonucleotide using only two wavelengths. The poor experimental RSCCs obtained from solution (i) are attributed to poor wavelength calibration of the !…”

“…The morphology of the crystal used in this experiment would have indeed been helpful in reducing these errors. Peterson et al (1996) demonstrated this clearly by measuring data at the f HH peak of the Br K edge with an aligned crystal of a brominated oligonucleotide and with the same crystal misaligned. Using information from only two wavelengths, they found that inclusion of the misaligned-crystal data gave an effectively uninterpretable map, whereas inclusion of the aligned-crystal data provided clearly better maps.…”

“…More recently, data measurement and analysis strategies for MAD have been investigated by Peterson et al (1996) using crystals of a brominated oligonucleotide.…”

Determination by MAD-DM of the structure of the DNA duplex d[ACGTACG(5-BrU)]2 at 1.46 Å and 100 K

“…The MAD method relies for its success on the careful measurement of anomalous differences (Peterson et al, 1996) and its success is highly dependent on the characteristics of the source. The third-generation source at Elettra, Trieste, Italy, has a stable, intense beam, and data collected there yielded MAD phases which, in combination with density modi®cation and histogram matching, yielding a very clear electron-density map by the methods described here.…”

Direct-method structure determination of the native azurin II protein using one-wavelength anomalous scattering data

“…However, MAD experiments have generally used three or more wavelengths to reach a least-squares determination of the phase angle, which puts increased demand on the highly scarce synchrotron radiation beamtime. In a recent effort to minimize this demand, several strategies for a MAD experiment have been explored and it is likely that at least in some cases the requirement could be reduced to data collection at two wavelengths (Peterson et at., 1996). Therefore, there have been great efforts to resolve the phase ambiguity arising from a one-wavelength anomalous scattering (OAS) technique without using additional diffraction data.…”