AbstractAPC/C is a multi-subunit complex that functions as a master regulator of cell division. It controls progression through the cell cycle by timely marking mitotic cyclins and other cell cycle regulatory proteins for degradation. The APC/C itself is regulated by the sequential action of its coactivator subunits CDC20 and CDH1, post-translational modifications, and its inhibitory binding partners EMI1 and the mitotic checkpoint complex.

摘要APC/C是一种多亚基复合物，可作为细胞分裂的主要调节因子。它通过及时标记有丝分裂细胞周期蛋白和其他细胞周期调节蛋白的降解来控制细胞周期的进展。APC/C本身受其共激活亚基CDC20和CDH1的顺序作用，翻译后修饰及其抑制性结合伴侣EMI1和有丝分裂检查点复合物的调节。

In this study, we took advantage of developments in cryo-electron microscopy to determine the structures of human APC/CCDH1:EMI1 and apo-APC/C at 2.9 Å and 3.2 Å resolution, respectively, providing insights into the regulation of APC/C activity. The high-resolution maps allow the unambiguous assignment of an α-helix to the N-terminus of CDH1 (CDH1α1) in the APC/CCDH1:EMI1 ternary complex.

在这项研究中，我们利用低温电子显微镜的发展来确定人类APC/CCDH1的结构：EMI1和apo APC/C的分辨率分别为2.9Å和3.2Å，为APC/C活性的调节提供了见解。高分辨率图谱允许将α-螺旋明确分配到APC/CCDH1:EMI1三元复合物中CDH1（CDH1α1）的N端。

We also identify a zinc-binding module in APC2 that confers structural stability to the complex, and we confirm the presence of zinc ions experimentally. Finally, due to the higher resolution and well defined density of these maps, we are able to build, aided by AlphaFold predictions, several intrinsically disordered regions in different APC/C subunits that likely play a role in proper APC/C assembly and regulation of its activity..

我们还确定了APC2中的锌结合模块，该模块赋予复合物结构稳定性，并且我们通过实验证实了锌离子的存在。最后，由于这些图谱具有更高的分辨率和明确的密度，我们能够在AlphaFold预测的帮助下，在不同的APC/C亚基中构建几个内在无序的区域，这些区域可能在正确的APC/C组装和调节其活性中发挥作用。。

IntroductionThe anaphase-promoting complex/cyclosome (APC/C) is a large multi-subunit E3 ubiqutin ligase that regulates transitions through the cell cycle by controlling the defined degradation of specific cell cycle regulators through the ubiquitin-proteasome system (UPS). APC/C activity and substrate selection are controlled at various levels to ensure the correct order of specific cell cycle events.

引言后期促进复合物/环体（APC/C）是一种大型多亚基E3泛素连接酶，通过泛素-蛋白酶体系统（UPS）控制特定细胞周期调节剂的降解来调节细胞周期的转变。APC/C活性和底物选择受到不同水平的控制，以确保特定细胞周期事件的正确顺序。

These regulatory mechanisms include the binding of cell cycle specific coactivator subunits (CDC20 and CDH1), reversible APC/C and coactivator phosphorylation, inhibitory complexes and proteins (for example the mitotic checkpoint complex (MCC) and early mitotic inhibitor 1 (EMI1)), and APC/C SUMOylation.

这些调节机制包括细胞周期特异性共激活因子亚基（CDC20和CDH1），可逆APC/C和共激活因子磷酸化，抑制复合物和蛋白质（例如有丝分裂检查点复合物（MCC）和早期有丝分裂抑制剂1（EMI1））和APC/C SUMO化。

APC/C function, mechanisms and structures have been reviewed extensively1,2,3,4.In mitosis the APC/C is activated by phosphorylation and by the binding of a coactivator subunit CDC20, to form the APC/CCDC20 complex that functions to ubiquitinate securin and cyclin B. APC/C-mediated destruction of both securin and cyclin B activates separase to cleave the cohesin ring, thereby triggering sister chromatid segregation and the onset of anaphase.

APC/C的功能，机制和结构已被广泛综述1,2,3,4。在有丝分裂中，APC/C通过磷酸化和共激活亚基CDC20的结合而被激活，形成APC/CCDC20复合物，其功能是泛素化securin和细胞周期蛋白B.APC/C介导的securin和细胞周期蛋白B的破坏激活分离酶以切割粘着蛋白环，从而触发姐妹染色单体分离和后期的开始。

During this transition, the APC/C and CDH1 are dephosphorylated, resulting in a switch from APC/CCDC20 to APC/CCDH1. In addition to mediating the metaphase to anaphase transition, the CDH1-activated APC/C regulates the events of cytokinesis and the entry into G1.The APC/C binds its substrates through destruction motifs or degrons: the D box, the ABBA motif and the KEN box5,6,7,8.

。除了介导中期到后期的转变外，CDH1激活的APC/C还调节胞质分裂和进入G1的事件。APC/C通过破坏基序或降解子结合其底物：D盒，ABBA基序和KEN盒5,6,7,8。

These motifs interact with their cognate receptors on two structurally related WD40-domain coactivator subunits (CDC20 in mitosis and CDH1 in G1) that function to recruit substrates to the APC/C. Control of different cell cycle phases by the APC/C is mediated.

这些基序与两个结构相关的WD40结构域共激活子亚基（有丝分裂中的CDC20和G1中的CDH1）上的同源受体相互作用，这些亚基的功能是将底物募集到APC/C。介导APC/C对不同细胞周期阶段的控制。

For this study, the APC/CCDH1:EMI1 ternary complex and cryo-EM grids were prepared as described previously19 (Supplementary Fig. 1). The cryo-EM map refinement work-flow, based on a multi-body approach, is detailed in Methods (Supplementary Figs. 1 and 2 and Supplementary Table 1). Three-dimensional classification generated eight 3D classes, two belonging to the APC/CCDH1:EMI1 ternary complex and two to the apo-APC/C state (that is without CDH1 and EMI1).

对于这项研究，如前所述19（补充图1）制备了APC/CCDH1:EMI1三元复合物和低温电磁网格。基于多体方法的cryo-EM map细化工作流程在方法（补充图1和2以及补充表1）中有详细说明。三维分类产生了八个3D类，其中两个属于APC/CCDH1:EMI1三元复合物，两个属于apo-APC/C状态（即没有CDH1和EMI1）。

The four remaining classes (classes 3-6; Supplementary Fig. 2) corresponded to incomplete APC/C assemblies. The APC/CCDH1:EMI1 ternary complex map was divided into three masks, with some overlap, for particle subtraction and focussed 3D refinement. This process greatly improved cryo-EM densities in the more peripheral and flexible regions of the map.

剩下的四个类别（类别3-6；补充图2）对应于不完整的APC/C组件。APC/CCDH1:EMI1三元复合图分为三个掩模，有一些重叠，用于粒子减法和聚焦3D细化。该过程极大地改善了地图更外围和更灵活区域的低温电磁密度。

Specifically, well defined cryo-EM density is visible for the flexible catalytic module comprising the APC2 C-terminal domain (APC2CTD), APC11 and the zinc-binding region (ZBR) of EMI1 (EMI1ZBR) (Fig. 1a, b and Supplementary Fig. 1c). Additionally, the APC7 homodimer, capping the TPR lobe, is well defined.

具体而言，对于包含APC2 C末端结构域（APC2CTD），APC11和EMI1（EMI1ZBR）的锌结合区（ZBR）的柔性催化模块，可以看到明确定义的低温电磁密度（图1a，b和补充图1c）。此外，覆盖TPR叶的APC7同型二聚体定义明确。

The current apo-APC/C structure at 3.2 Å supersedes the phosphorylated and unphosphorylated apo-APC/C maps at 3.4 Å and 3.8 Å, respectively27.As outlined in Supplementary Table 2, these higher resolution maps allowed marked improvements to regions of the structure compared to the previous 3.6 Å map19 (PDB 4UI9) according to three main criteria – (i) rebuilding of poorly defined loops; (ii) the fitting of side chains and hence residue assignment to previously built poly-Ala regions; (iii) de novo building of specific regions of the molecule.

目前3.2Å的apo-APC/C结构分别取代了3.4Å和3.8Å的磷酸化和未磷酸化apo-APC/C图27。如补充表2中所述，根据三个主要标准，与之前的3.6Åmap19（PDB 4UI9）相比，这些更高分辨率的图可以显着改善结构区域—（i）重建定义不明确的环；（ii）侧链的拟合，从而将残基分配给先前构建的poly-Ala区域；（iii）从头构建分子的特定区域。

Four rebuilt or de novo built regions have biological implications and are discussed in more detail below.An N-terminal α-helix of m.

四个重建或从头建造的区域具有生物学意义，下面将进行更详细的讨论。m的N端α-螺旋。

Data availability

数据可用性

PDB coordinates generated in this study have been deposited with RCSB under accession codes 8PKP and 9GAW Cryo-EM maps generated in this study have been deposited with EMDB under accession codes EMD-17751, EMD-51190, EMD-13931), EMD-13932), EMD-13933 ([https://www.ebi.ac.uk/emdb/EMD-13933]), EMD-51070 ([https://www.ebi.ac.uk/emdb/EMD-51070]), EMD-19711 ([https://www.ebi.ac.uk/emdb/EMD-19711].

本研究中生成的PDB坐标已保存在RCSB中，登录号为8PKP，本研究中生成的9GAW低温电磁图已保存在EMDB中，登录号为EMD-17751，EMD-51190，EMD-13931），EMD-13932），EMD-13933([https://www.ebi.ac.uk/emdb/EMD-13933])，EMD-51070([https://www.ebi.ac.uk/emdb/EMD-51070])，EMD-19711([https://www.ebi.ac.uk/emdb/EMD-19711]。

Previously published protein coordinates used in study: 4UI9 and 5G05. Accession numbers are also listed in Supplementary Table 1. Source data are provided with this paper. Correspondence and requests for materials should be addressed to David Barford. Source data are provided with this paper..

先前公布的研究中使用的蛋白质坐标：4UI9和5G05。补充表1中也列出了登录号。本文提供了源数据。信件和材料要求应寄给David Barford。本文提供了源数据。。

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Download referencesAcknowledgementsWe are grateful to the LMB EM Facility for help with the EM data collection, J. Grimmett, T. Darling and I. Clayson for computing, and J. Shi for help with insect cell expression. We thank Stanislau Yatskevich for reagents and A. Andreeva for advice on the APC2ZBM structure.

下载参考文献致谢我们感谢LMB EM设施对EM数据收集的帮助，J.Grimmett，T.Darling和I.Clayson的计算，以及J.Shi对昆虫细胞表达的帮助。我们感谢Stanislau Yatskevich提供的试剂和A。Andreeva就APC2ZBM结构提出建议。

This work was supported by grants of the Swiss National Science Foundation (310030_185235 and MSGI3_211581) (A.B.) and the Swiss Cancer League (KFS-5453-08-2021) (A.B.) and by UKRI/Medical Research Council MC_UP_1201/6 (D.B.) and Cancer Research UK C576/A14109 (D.B.).Author informationAuthor notesThese authors contributed equally: Anna Höfler, Jun Yu.Authors and AffiliationsDepartment of Molecular and Cellular Biology, University of Geneva, Geneva, SwitzerlandAnna Höfler, Jun Yu & Andreas BolandMRC Laboratory of Molecular Biology, Cambridge, CB2 0QH, UKJing Yang, Ziguo Zhang, Leifu Chang, Stephen H.

这项工作得到了瑞士国家科学基金会（310030'U 185235和MSGI3'U 211581）（A.B.）和瑞士癌症联盟（KFS-5453-08-2021）（A.B.）以及UKRI/医学研究委员会MC\U UP\U 1201/6（D.B.）和英国癌症研究C576/A14109（D.B.）的资助。作者信息作者注意到这些作者做出了同样的贡献：Anna Höfler，Jun Yu。作者和附属机构日内瓦大学分子与细胞生物学系，瑞士赫尔南纳·Höfler，Jun Yu＆Andreas BolandMRC分子生物学实验室，剑桥，CB2 0QH，UKJing Yang，Ziguo Zhang，Leifu Chang，Stephen H。

McLaughlin & David BarfordIon Beam Centre, Advanced Technology Institute, University of Surrey, Guildford, Surrey, GU2 7XH, UKGeoffrey W. GrimeDepartment of Biochemistry, University of Oxford, Dorothy Crowfoot Hodgkin Building, South Parks Road, Oxford, OX1 3QU, UKElspeth F. GarmanAuthorsAnna HöflerView author publicationsYou can also search for this author in.

McLaughlin＆David BarfordIon光束中心，萨里大学高级技术研究所，吉尔福德，萨里，GU2 7XH，英国杰弗里·W·格里姆生物化学系，牛津大学，多萝西·克劳福德·霍奇金大厦，牛津大学南帕克斯路，OX1 3QU，UKElspeth F.GarmanAuthorsAnna HöflerView作者出版物您也可以在中搜索这位作者。

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PubMed Google ScholarContributionsThe project has been conceived by D.B, A.B. and L.C. Experiments were performed by A.H., J.Yu, J.Yang., Z.Z., L.C., S.H.M, G.W.G., E.F.G., A.B., D.B. The paper was written by D.B., A.B., A.H., J.Yu, with input from all other authors.Corresponding authorsCorrespondence to.

。，Z、 Z.，L.C.，S.H.M，G.W.G.，E.F.G.，A.B.，D.B。论文由D.B.，A.B.，A.H.，J.Yu撰写，所有其他作者均提供了意见。。

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Reprints and permissionsAbout this articleCite this articleHöfler, A., Yu, J., Yang, J. et al. Cryo-EM structures of apo-APC/C and APC/CCDH1:EMI1 complexes provide insights into APC/C regulation.

转载和许可本文引用本文Höfler，A.，Yu，J.，Yang，J。等人。载脂蛋白APC/C和APC/CCDH1的低温电磁结构：EMI1复合物提供了对APC/C调控的见解。

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