Applying an Empirical Hydropathic Forcefield in Refinement May Improve Low-Resolution Protein X-Ray Crystal Structures

BackgroundThe quality of X-ray crystallographic models for biomacromolecules refined from data obtained at high-resolution is assured by the data itself. However, at low-resolution, >3.0 Å, additional information is supplied by a forcefield coupled with an associated refinement protocol. These resulting structures are often of lower quality and thus unsuitable for downstream activities like structure-based drug discovery.
MethodologyAn X-ray crystallography refinement protocol that enhances standard methodology by incorporating energy terms from the HINT (Hydropathic INTeractions) empirical forcefield is described. This protocol was tested by refining synthetic low-resolution structural data derived from 25 diverse high-resolution structures, and referencing the resulting models to these structures. The models were also evaluated with global structural quality metrics, e.g., Ramachandran score and MolProbity clashscore. Three additional structures, for which only low-resolution data are available, were also re-refined with this methodology.
ResultsThe enhanced refinement protocol is most beneficial for reflection data at resolutions of 3.0 Å or worse. At the low-resolution limit, ≥4.0 Å, the new protocol generated models with Cα positions that have RMSDs that are 0.18 Å more similar to the reference high-resolution structure, Ramachandran scores improved by 13%, and clashscores improved by 51%, all in comparison to models generated with the standard refinement protocol. The hydropathic forcefield terms are at least as effective as Coulombic electrostatic terms in maintaining polar interaction networks, and significantly more effective in maintaining hydrophobic networks, as synthetic resolution is decremented. Even at resolutions ≥4.0 Å, these latter networks are generally native-like, as measured with a hydropathic interactions scoring tool.

【背景】利用高分辨率实验数据解析得到的生物大分子X射线晶体学模型，其质量可由实验数据本身保障。但当分辨率低于3.0 Å（即低分辨率场景）时，则需结合力场与配套的精修协议补充额外信息。此类方法得到的结构通常质量欠佳，因此不适用于基于结构的药物发现等下游科研活动。

【方法】本文提出一种增强型X射线晶体学精修协议，其通过引入HINT（亲水相互作用，Hydropathic INTeractions）经验力场的能量项优化标准精修流程。本研究使用25组不同来源的高分辨率结构生成模拟低分辨率结构数据，以此开展精修测试，并将得到的模型与原始高分辨率结构进行比对。同时采用全局结构质量评估指标对模型进行校验，例如拉马昌德兰评分（Ramachandran score）与MolProbity冲突评分。此外，本研究还使用该方法对仅具备低分辨率实验数据的3组额外结构进行了二次精修。

【结果】该增强型精修协议对分辨率不高于3.0 Å的衍射数据优化效果最为显著。在低分辨率极限（≥4.0 Å）场景下，相较于标准精修协议得到的模型，新协议生成的模型中Cα原子位置的均方根偏差（RMSD）较参考高分辨率结构低0.18 Å，拉马昌德兰评分提升13%，冲突评分改善51%。随着模拟分辨率降低，该亲水力场项在维持极性相互作用网络方面的效果至少与库仑静电项相当，而在维持疏水相互作用网络方面则显著更优。即使在分辨率≥4.0 Å的场景下，通过亲水相互作用评分工具评估，此类疏水网络整体仍接近天然状态。