The structural ensemble of a Holliday junction determined by X-ray scattering interference.

The DNA four-way (Holliday) junction is the central intermediate of genetic recombination, yet key aspects of its conformational and thermodynamic properties remain unclear. While multiple experimental approaches have been used to characterize the canonical X-shape conformers under specific ionic conditions, the complete conformational ensemble of this motif, especially at low ionic conditions, remains largely undetermined. In line with previous studies, our single-molecule Förster resonance energy transfer (smFRET) measurements of junction dynamics revealed transitions between two states under high salt conditions, but smFRET could not determine whether there are fast and unresolvable transitions between distinct conformations or a broad ensemble of related states under low and intermediate salt conditions. We therefore used an emerging technique, X-ray scattering interferometry (XSI), to directly probe the conformational ensemble of the Holliday junction across a wide range of ionic conditions. Our results demonstrated that the four-way junction adopts an out-of-plane geometry under low ionic conditions and revealed a conformational state at intermediate ionic conditions previously undetected by other methods. Our results provide critical information to build toward a full description of the conformational landscape of the Holliday junction and underscore the utility of XSI for probing conformational ensembles under a wide range of solution conditions.