Abstact

Despite the theoretical advantages of phosphorus single-wavelength anomalous diffraction (P-SAD) for nucleic acid phasing, its application remains limited due to high atomic displacement parameters and an unfavourable ratio of unique reflections to anomalous scatterers. In this study, we report the crystal structure of an RNA complex composed of four strands, which was solved by experimental phasing after AlphaFold3 failed to produce reliable models. Bromine single-wavelength anomalous diffraction (Br-SAD) data were collected at 0.916 Å on beamline I04 at Diamond Light Source, while phosphorus anomalous data were obtained at 3.024 Å on beamline I23. The structure was successfully phased using bromine anomalous scattering, and phosphorus anomalous peaks corroborated the backbone positions and validated the model. Attempts to phase the structure directly from phosphorus data failed, consistent with theoretical predictions that successful SAD phasing requires a significantly higher reflection-to-scatterer ratio. The final models reveal an RNA complex stabilized by Watson-Crick and Hoogsteen base pairing, forming a pseudo-helical complex instead of the anticipated hairpin stem-loop, likely reflecting crystallization artefacts. This work demonstrates the complementary use of bromine and phosphorus anomalous signals in RNA crystallography.