目的·通过负染色及透射电子显微镜（电镜）技术分析非经典型多梳抑制复合物1.6（polycomb repressive complex 1.6，PRC1.6）的蛋白结构，获得人源PRC1.6七元复合物的三维轮廓信息。方法·将PRC1.6复合物中7个组分基因RNF2、PCGF6、RYBP、L3MBTL2、CBX3、E2F6和TFDP1分别克隆至N端带有6×His-3×Flag标签的pMLink载体中，利用聚乙烯亚胺瞬时转染的方式在悬浮培养的哺乳动物细胞Expi293F中表达PRC1.6七元复合物，依次使用anti-DYKDDDDK标签亲合树脂、分子筛Superdex 200 Increase 10/300 GL（凝胶过滤层析）和甘油密度梯度离心分离纯化PRC1.6复合物；通过液相色谱-串联质谱法（liquid chromatography-tandem mass spectrometry，LC-MS/MS）确认PRC1.6七元复合物组分；利用泛素化活性实验和电泳迁移率变动分析（electrophoretic mobility shift assay，EMSA）对复合物的体外泛素化活性以及与核小体结合亲和力进行验证；使用乙酸双氧铀进行样品制备并通过透射电镜观察蛋白样品，随后通过单颗粒重构技术对PRC1.6复合物的三维结构信息进行分析；利用UCSF Chimera软件将蛋白质结构数据库（Protein Data Bank，PDB）中的目的蛋白相关原子结构模型与重构获得的PRC1.6电子密度图进行拟合，预测PRC1.6七元复合物中各亚基的定位。结果·通过真核表达、亲和层析、凝胶过滤层析和甘油密度梯度离心，以及LC-MS/MS验证，获得了纯度较高、均一性较好的PRC1.6七元复合物，且泛素化活性实验和EMSA发现该复合物在体外具有泛素化活性和核小体结合亲和力。利用负染色、透射电镜和单颗粒重构技术初步解析了分辨率约为15.2 ?（1 ?=10^-10 m）的PRC1.6七元复合物的三维结构，将已有RNF2、PCGF6、RYBP、L3MBTL2、CBX3和DP1部分氨基酸序列的原子模型以及E2F6的AlphaFold2预测结构与重构获得的电子密度图进行匹配，初步确认了7个亚基在PRC1.6复合物三维结构中的定位情况。结论·使用负染色和透射电镜，以及单颗粒重构技术搭建了人源PRC1.6七元复合物的三维结构模型。

Objective ·To analyse the structure of non-canonical polycomb repressive complex 1.6 (PRC1.6) by negative staining and transmission electron microscopy (TEM), and obtain the three-dimensional (3D) profile information of human PRC1.6 heptameric complex. Methods ·Seven PRC1.6 components, RNF2, PCGF6, RYBP, L3MBTL2, CBX3, E2F6, and TFDP1, were cloned into the pMLink vector with a 6×His-3×Flag tag at the N-terminus, respectively. The proteins were expressed in Expi293F cells grown in suspension cultures by using transfection with polyethylenimine. The tagged proteins were isolated via affinity purification with anti-DYKDDDDK G1 affinity resin, followed by gel filtration chromatography with Superdex 200 Increase 10/300 GL and glycerol density gradient centrifugation. The components of the PRC1.6 heptameric complex were confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The in vitro ubiquitination activity and nucleosome-binding affinity of the purified heptameric complex were verified by the ubiquitination activity assay and the electrophoretic mobility shift assay (EMSA). The protein samples were stained by uranyl acetate and observed by TEM. The 3D information of the PRC1.6 complex was studied by single particle analysis. To predict the localization of the seven components within the structure model of PRC1.6 complex, the structure models of proteins in Protein Data Bank (PDB) were docked into the electron density map of PRC1.6 complex by using UCSF Chimera software. Results ·The PRC1.6 complex with high purity and good homogeneity was obtained by eukaryotic expression, affinity purification, gel filtration chromatography and glycerol density gradient centrifugation, and confirmed as the heptameric complex by LC-MS/MS. The purified proteins showed ubiquitination activity and nucleosome-binding affinity in vitro. The 3D structure of the PRC1.6 heptameric complex with a resolution of 15.2 ? (1 ?=10^-10 m) was preliminarily resolved by negative staining, TEM, and single particle analysis. The available structure models of RNF2, PCGF6, RYBP, L3MBTL2, CBX3, and DP1 proteins, as well as the predicted E2F6 structure by AlphaFold2, were docked into the reconstructed density map of PRC1.6 complex. The position of each component in the complex was preliminarily confirmed. Conclusion ·The 3D structural model of the human PRC1.6 heptameric complex is obtained by negative staining, TEM, and single particle analysis.