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.

中文翻译：

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通过X射线散射干涉确定的霍利迪结的结构整体。

DNA四向（霍利迪）连接是基因重组的核心中间产物，但其构象和热力学性质的关键方面仍不清楚。虽然已使用多种实验方法来表征特定离子条件下的典型X形构象体，但该基序的完整构象整体（尤其是在低离子条件下）仍未定。与以前的研究一致，我们的结合动力学单分子福斯特共振能量转移（smFRET）测量揭示了在高盐条件下两种状态之间的转变，但是smFRET无法确定不同构象或宽整体之间是否存在快速且不可分辨的转变在中低盐条件下的相关状态 因此，我们使用了一种新兴技术，X射线散射干涉仪（XSI），可在各种离子条件下直接探测霍利迪结的构象整体。我们的结果表明，四向结在低离子条件下采用平面外几何形状，并在中等离子条件下显示出构象状态，该状态以前是其他方法无法检测到的。我们的结果提供了关键信息，可帮助您完整描述霍利迪交汇处的构象景观，并强调了XSI在广泛解决方案条件下探测构象集合的实用性。我们的结果表明，四向结在低离子条件下采用平面外几何形状，并在中等离子条件下显示出构象状态，该状态以前是其他方法无法检测到的。我们的结果提供了关键信息，可帮助您完整描述霍利迪交汇处的构象景观，并强调了XSI在广泛的溶液条件下探测构象集合的实用性。我们的结果表明，四向结在低离子条件下采用平面外几何形状，并在中等离子条件下显示出构象状态，该状态以前是其他方法无法检测到的。我们的结果提供了关键信息，可帮助您完整描述霍利迪交汇处的构象景观，并强调了XSI在广泛解决方案条件下探测构象集合的实用性。