Polarization-dependence of anomalous scattering in brominated DNA and RNA molecules, and importance of crystal orientation in single- and multiple-wavelength anomalous diffraction phasing

Abstract

In this paper the anisotropy of anomalous scattering at the Br K-absorption edge in brominated nucleotides is investigated, and it is shown that this effect can give rise to a marked directional dependence of the anomalous signal strength in X-ray diffraction data. This implies that choosing the correct orientation for crystals of such molecules can be a crucial determinant of success or failure when using single-and multiple-wavelength anomalous diffraction (SAD or MAD) methods to solve their structure. In particular, polarized absorption spectra on an oriented crystal of a brominated DNA molecule were measured, and were used to determine the orientation that yields a maximum anomalous signal in the diffraction data. Out of several SAD data sets, only those collected at or near that optimal orientation allowed interpretable electron density maps to be obtained. The findings of this study have implications for instrumental choices in experimental stations at synchrotron beamlines, as well as for the development of data collection strategy programs.

Exploiting the anisotropy of anomalous scattering boosts the phasing power of SAD and MAD experiments

“…compound can be expected to be very similar to that of brominated nucleotides (Sanishvili et al, 2007). This was confirmed by a series of polarized absorption spectra recorded on triclinic crystals of p-bromobenzamide, in which all the C-Br bonds in the unit cell are parallel (Schiltz et al, unpublished results).…”

“…The work described here was preceded by extensive developments of computer code for simulating data affected by AAS and analysing them for the purpose of phase determination (Schiltz & Bricogne, unpublished results). Experimental investigations of AAS effects in macromolecules at the level of polarized absorption spectra were reported by Bricogne et al (2005) and an example of their impact on the success or failure of a SAD phasing experiment was given by Sanishvili et al (2007). Here, we finally tackle the central topic of incorporating AAS effects into the general process of macromolecular phasing.…”

“…Green curves: along the direction w (perpendicular to the nucleobase ring). Data from Sanishvili et al (2007).…”

“…4). We have previously shown (Sanishvili et al, 2007) that there is a pronounced directional dependence of the anomalous signal strength in X-ray diffraction data collected at the Br K absorption edge from these crystals and that choosing the correct orientation for crystals of such molecules can be a crucial determinant of success or failure when using SAD or MAD methods to solve their structure. Here, we show that beyond the directional dependence of the overall anomalous signal, the anomalous scattering strength of each Br site in the , e y , e z ) which sets e x parallel to a and e z parallel to c*.…”

Stacking of the mutagenic base analogue 5-bromouracil: energy landscapes of pyrimidine dimers in gas phase and water

“…1, right). [17][18][19] As well as having only two hydrogen bonds (compared to three in the enolised mispair), the wobble pair's incorrect stereochemistry appears to make it an unlikely candidate for mutagenic replication. However, it may be stabilised against enzymatic repair (and hence be mutagenic) if it takes part in stronger base stacking interactions than the corresponding T-G or U-G wobble pairs, due to the bromine substituent.…”