Phosphorus SAD Phasing for Nucleic Acid Structures: Limitations and Potential

Abstract

Phasing of nucleic acid crystal diffraction data using the anomalous signal of phosphorus, P-SAD, at Cuk α wavelength has been previously demonstrated using Z-DNA. Since the original work on P-SAD with Z-DNA there has been, with a notable exception, a conspicuous absence of applications of the technique to additional nucleic acid crystal structures. We have reproduced the P-SAD phasing of Z-DNA using a rotating-anode source and have attempted to phase a variety of nucleic acid crystals using P-SAD without success. A comparison of P-SAD using Z-DNA and a representative nucleic acid, the Dickerson-Drew dodecamer, is presented along with a S-SAD using only two sulfurs to phase a 2'-thio modified DNA decamer. A theoretical explanation for the limitation of P-SAD applied to nucleic acids is presented to show that the relatively high atomic displacement parameter of phosphorus in the nucleic acid backbone is responsible for the lack of success in applying P-SAD to nucleic acid diffraction data.

Native de novo structural determinations of non-canonical nucleic acid motifs by X-ray crystallography at long wavelengths

“…Here because of the small unit cell, the number of reflections is low, while the number of phosphorous atoms is high (∼1 per nucleotide). Additionally, the apparent mobility of the phosphates in DNA/RNA structures, either static or dynamic, conspires to reduce the strength of anomalous scattering ( 39 ). In this analysis, there was an insufficient P-SAD anomalous contribution which required us to combine the use of both intrinsic (P-SAD) and extrinsic (Br-SAD) anomalous scattering for a successful structural determination (Figure 4G – J ).…”

“…Currently, automated pipelines for phasing and model building are not as robust for nucleic acids as for proteins. This is particularly relevant to advance the effective incorporation of P-SAD methods ( 39 , 53 ) in phase determination. However, at this stage, we have shown the general utility of using intrinsic scattering to aid in the identification of metal ions, such as K + .…”

Solid-phase synthesis and structural characterisation of phosphoroselenolate-modified DNA: a backbone analogue which does not impose conformational bias and facilitates SAD X-ray crystallography

“…Furthermore, the furanose pseudorotation angles of the most common modication, 2 0 -SeMe, typically results in conformational steering towards an A-form duplex. Using the related 2 0 -SMe analogue, such conformational restrictions were not observed and both A-and B-DNA duplex crystal structures were solved 82,83 although longer collection times (and concomitant DNA damage) are associated with the weaker anomalous scattering from sulfur at the CuK a wavelength. Furthermore, both analogues interfere with minor groove binding interactions.…”