Structural heterogeneity of nucleosomes in functional chromosomes is unknown. Here, we devise the template-, reference- and selection-free (TRSF) cryo-EM pipeline to simultaneously reconstruct cryo-EM structures of protein complexes from interphase or metaphase chromosomes. The reconstructed interphase and metaphase nucleosome structures are on average indistinguishable from canonical nucleosome structures, despite DNA sequence heterogeneity, cell-cycle-specific posttranslational modifications, and interacting proteins. Nucleosome structures determined by a decoy-classifying method and structure variability analyses reveal the nucleosome structural variations in linker DNA, histone tails, and nucleosome core particle configurations, suggesting that the opening of linker DNA, which is correlated with H2A C-terminal tail positioning, is suppressed in chromosomes. High-resolution (3.4–3.5 Å) nucleosome structures indicate DNA-sequence-independent stabilization of superhelical locations ±0–1 and ±3.5–4.5. The linker histone H1.8 preferentially binds to metaphase chromatin, from which chromatosome cryo-EM structures with H1.8 at the on-dyad position are reconstituted. This study presents the structural characteristics of nucleosomes in chromosomes.

功能染色体中核小体的结构异质性尚不清楚。在这里，我们设计了无模板、无参考和无选择（TRSF）冷冻电镜管道，以同时重建来自间期或中期染色体的蛋白质复合物的冷冻电镜结构。尽管存在 DNA 序列异质性、细胞周期特异性翻译后修饰和相互作用的蛋白质，但重建的间期和中期核小体结构与典型核小体结构平均无法区分。通过诱饵分类方法和结构变异性分析确定的核小体结构揭示了接头 DNA、组蛋白尾部和核小体核心颗粒构型的核小体结构变化，表明接头 DNA 的打开与 H2A C 末端尾部定位相关，在染色体中被抑制。高分辨率 (3.4–3.5 Å) 核小体结构表明超螺旋位置 ±0–1 和 ±3.5–4.5 具有独立于 DNA 序列的稳定性。连接组蛋白 H1.8 优先与中期染色质结合，由此重建在二联体位置具有 H1.8 的染色体冷冻电镜结构。这项研究展示了染色体中核小体的结构特征。