School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore.
NTU Institute of Structural Biology, Nanyang Technological University, 59 Nanyang Drive, 636921, Singapore.
Nucleic Acids Res. 2021 May 21;49(9):e52. doi: 10.1093/nar/gkab070.
Structural characterization of chromatin is challenging due to conformational and compositional heterogeneity in vivo and dynamic properties that limit achievable resolution in vitro. Although the maximum resolution for solving structures of large macromolecular assemblies by electron microscopy has recently undergone profound increases, X-ray crystallographic approaches may still offer advantages for certain systems. One such system is compact chromatin, wherein the crystalline state recapitulates the crowded molecular environment within the nucleus. Here we show that nucleosomal constructs with cohesive-ended DNA can be designed that assemble into different types of circular configurations or continuous fibers extending throughout crystals. We demonstrate the utility of the method for characterizing nucleosome compaction and linker histone binding at near-atomic resolution but also advance its application for tackling further problems in chromatin structural biology and for generating novel types of DNA nanostructures. We provide a library of cohesive-ended DNA fragment expression constructs and a strategy for engineering DNA-based nanomaterials with a seemingly vast potential variety of architectures and histone chemistries.
由于体内构象和组成的异质性以及体外限制可达到分辨率的动态特性，染色质的结构表征具有挑战性。尽管最近电子显微镜解决大型大分子组装结构的最大分辨率有了深刻的提高，但X 射线晶体学方法对于某些系统可能仍然具有优势。一个这样的系统是紧凑的染色质，其中晶体状态再现了核内拥挤的分子环境。在这里，我们展示了具有粘性末端 DNA 的核小体构建体可以被设计成组装成不同类型的圆形构象或连续纤维，延伸到整个晶体中。我们证明了该方法在接近原子分辨率下用于表征核小体紧缩和连接组蛋白结合的用途，但也推进了其在解决染色质结构生物学中进一步问题以及生成新型 DNA 纳米结构方面的应用。我们提供了一个粘性末端 DNA 片段表达构建体库，并提供了一种用于工程化基于 DNA 的纳米材料的策略，该策略具有看似广泛的各种架构和组蛋白化学的潜力。
