Phosphorus SAD Phasing for Nucleic Acid Structures: Limitations and Potential

Joel Harp,Pradeep Pallan,Martin Egli

doi:10.3390/cryst6100125

Abstract

Phasing of nucleic acid crystal diffraction data using the anomalous signal of phosphorus, P-SAD, at Cukα 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.

Highlights

Anomalous dispersion phasing is an attractive approach to solving the phase problem in crystallography of biological macromolecules
We have explored the possibility of P-single-wavelength anomalous dispersion (SAD) phasing for several DNA and RNA oligonucleotides of known or unknown crystal structure using data collected on an in-house source with copper Kα radiation
Data were collected to a maximum resolution of 0.95Å matching the resolution of data used by Adamiak and Dauter [52]

Summary

Introduction

Anomalous dispersion phasing is an attractive approach to solving the phase problem in crystallography of biological macromolecules. Experimental phases determined using anomalous dispersion techniques are capable of producing interpretable electron density maps without knowledge of the sequence of the protein or nucleic acid. The ability to exploit these techniques obviates the requirement for previously determined structures of similar conformation for molecular replacement phasing. The presence of a tractable anomalous signal depends on the presence of a suitable anomalous scatterer and the ability to collect diffraction data to adequate precision to extract that signal. The anomalous scatterer is an artificial addition to the structure to be determined. The anomalous scatterer is an atom naturally occurring in the molecule whose structure is to be determined precluding any question of isomorphism

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Results

Conclusion