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Cryo-EM structures of Smc5/6 in multiple states reveal its assembly and functional mechanisms

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Abstract

Smc5/6 is a member of the eukaryotic structural maintenance of chromosomes (SMC) family of complexes with important roles in genome maintenance and viral restriction. However, limited structural understanding of Smc5/6 hinders the elucidation of its diverse functions. Here, we report cryo-EM structures of the budding yeast Smc5/6 complex in eight-subunit, six-subunit and five-subunit states. Structural maps throughout the entire length of these complexes reveal modularity and key elements in complex assembly. We show that the non-SMC element (Nse)2 subunit supports the overall shape of the complex and uses a wedge motif to aid the stability and function of the complex. The Nse6 subunit features a flexible hook region for attachment to the Smc5 and Smc6 arm regions, contributing to the DNA repair roles of the complex. Our results also suggest a structural basis for the opposite effects of the Nse1–3–4 and Nse5–6 subcomplexes in regulating Smc5/6 ATPase activity. Collectively, our integrated structural and functional data provide a framework for understanding Smc5/6 assembly and function.

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Fig. 1: Cryo-EM structure of the eight-subunit Smc5/6 complex.
Fig. 2: Structures of six-subunit and five-subunit Smc5/6 and comparison with that of the Smc5/6-8mer.
Fig. 3: Nse2 contributes to an elongated conformation of Smc5/6 and its roles in cells.
Fig. 4: The Nse6 hook region bridges the joints of Smc5 and Smc6 and aids in Smc5/6 function.
Fig. 5: A model for structural changes and associated ATPase activities of three states of Smc5/6.

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Data availability

The cryo-EM maps and atomic coordinates have been deposited with the Electron Microscopy Data Bank and the PDB with the following accession codes: EMD-34025 (PDB: 7YQH, Smc5/6-8mer-overall), EMD-34953 (PDB: 8HQS, Smc5/6-8mer-head), EMD-37587 (PDB: 8WJO, Smc5/6-8mer-arm), EMD-33914 (PDB: 7YLM, Smc5/6-8mer-hinge), EMD-35116 (PDB: 8I13, Smc5/6-6mer-overall), EMD-37586 (PDB: 8WJN, Smc5/6-6mer-head), EMD-35128 (PDB: 8I21, Smc5/6-6mer-arm), EMD-37584 (PDB: 8WJL, Smc5/6-6mer-hinge), EMD-35187 (PDB: 8I4X, Smc5/6-5mer-overall), EMD-35186 (PDB: 8I4W, Smc5/6-5mer-head), EMD-35185 (PDB: 8I4V, Smc5/6-5mer-arm), EMD-35184 (PDB: 8I4U, Smc5/6-5mer-hinge) and EMD-33927 (PDB: 7YMD, Nse1–3–4). Structures used for structural comparisons/analyses have the following accession codes from the PDB: 5mg8, 3htk, 7lto, 7ogg, 7tve, 7dg2, 7qcd, 7ogt, 6yvu and 7nyy. The AlphaFold identifiers used for model building include AF-Q08204-F1 (Smc5), AF-Q12749-F1 (Smc6), AF-Q07913-F1 (Nse1), AF-P38632-F1 (Nse2), AF-Q05541-F1 (Nse3), AF-P43124-F1 (Nse4), AF-Q03718-F1 (Nse5) and AF-P40026-F1 (Nse6). Source data are provided with this paper.

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Acknowledgements

We thank C. Chen at Institut Pasteur of Shanghai, CAS for generous providing us with strain S288C; H. Zhao, Y. Ren, W. Zhan and Q. Mao at Fudan University (Fenglin Campus) for their assistance with cryo-EM data collection; the staff members of the scientific core facility in Institut Pasteur of Shanghai, CAS for their assistance with EM data analysis; the staff members of the Large-scale Protein Preparation System at the National Facility for Protein Science in Shanghai (NFPS), Shanghai Advanced Research Institute, CAS for providing technical support and assistance in data collection and analysis using Octet RED 96. We acknowledge a recently published study describing similar structures for parts of the Smc5/6-8mer in the presence of ATP and of the Smc5/6-5mer in the absence of ATP53. This work was supported by the grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB29010205, to L.W.); the National Key R&D Program of China (2022YFA1303600, to L.W.); Shanghai Municipal Science and Technology Major Project (2019SHZDZX02, to L.W.); NIGMS grant R35GM145260 and MSKCC BRIA grant to X. Zhao. This research was funded in part through the National Institutes of Health/National Cancer Institute Cancer Center Support Grant P30 CA008748.

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Author notes

  1. These authors contributed equally: Qian Li, Jun Zhang, Cory Haluska, **ang Zhang.

Authors and Affiliations

  1. The Center for Microbes, Development and Health, CAS Key Laboratory of Molecular Virology and Immunology, Shanghai Institute of Immunity and Infection, Chinese Academy of Sciences,University of Chinese Academy of Sciences, Shanghai, China

    Qian Li, Jun Zhang, Zhaoning Wang, Duo **, Tong Cheng, Hongxia Wang, Yuan Tian & Lanfeng Wang

  2. College of Life Sciences, University of Chinese Academy of Sciences, Bei**g, China

    Qian Li, Jun Zhang, Lei Wang, Zhaoning Wang, Duo **, Tong Cheng, **angxi Wang & Lanfeng Wang

  3. Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Cory Haluska & **aolan Zhao

  4. Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS) and Shanghai Institute of Infectious Disease and Biosecurity, Shanghai Fifth People’s Hospital, Shanghai Public Health Clinical Center, Institutes of Biomedical Sciences, School of Basic Medical Sciences,Fudan University, Shanghai, China

    **ang Zhang, Lei Sun & Zhenguo Chen

  5. CAS Key Laboratory of Infection and Immunity, National Laboratory of Macromolecules, Institute of Biophysics,Chinese Academy of Sciences, Bei**g, China

    Lei Wang & **angxi Wang

  6. National Center for Protein Science Shanghai, Shanghai Advanced Research Institute,Chinese Academy of Sciences, Shanghai, China

    Guangfeng Liu

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  1. Qian Li
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  2. Jun Zhang
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Contributions

L.W. and X. Zhao conceived and coordinated this project. Q.L. and J.Z. prepared protein samples, collected cryo-EM data, built structural models and conducted ATPase assay and DNA binding assay. C.H. carried out in vivo experiments. X. Zhang, L.S. and Z.C. participated in cryo-EM data collection, data analysis and model building. L.W. and X.W. helped with structure determination. G.L. and J.Z. collected and analyzed SAXS data. D.J., Z.W., T.C., H.W. and Y.T. assisted with sample preparation and data presentation. Q.L., J.Z., L.W. and X. Zhao wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to **aolan Zhao, Zhenguo Chen or Lanfeng Wang.

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Nature Structural & Molecular Biology thanks Damien D’Amours and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Sara Osman was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Cryo-EM structure determination of the Smc5/6-8mer.

(a) A representative micrograph of the Smc5/6-8mer cryo-EM samples. The microscopic examination has been repeated at least three samples independently with similar results. (b) Representative 2D classifications of the Smc5/6-8mer. (c) The workflow of cryo-EM image processing and map reconstruction.

Extended Data Fig. 2 Detailed cartoon presentations and local density maps of the Smc5/6-8mer complex.

(a) Cartoon presentation of individual subunit of the Smc5/6-8mer complex. Each subunit is highlighted in Smc5/6-8mer for clarity. (b) The local cryo-EM maps of Nse5, Nse6 and the Smc6 head domain in a focused refinement at an overall resolution of 3.2 Å in Smc5/6-8mer.

Extended Data Fig. 3 Structural analysis of the Nse2-wedge and the α1 region of Nse6-hook.

(a) The Nse2-wedge region seen in Smc5/6-8mer structure as described in this work (magenta) and as reported for the Nse2 structure (grey; PDB: 3HTK) (top). Density maps of both the Nse2-wedge and the interacting Smc6 helix at multiple surface volume levels, with the interface marked by black oval (bottom). (b) The density map of the Nse6-hook region at multiple surface volume levels, with the α1 region marked by red oval. (c) Secondary structure prediction of Nse6 using GOR IV (top) and PSIPRED tools (bottom). The α1 region is marked by a red rectangle. (d) Representative cartoon of the α1 region of Nse6-hook predicted using AlphaFold2.

Extended Data Fig. 4 Cryo-EM structure determination of the Nse1-3-4 subcomplex.

(a) A SEC profile of Nse1-3-4 subcomplex (left) with the peak fraction evaluated by SDS-PAGE (right). The sample purification has been repeated at least three times independently with similar results. (b) A representative micrograph of the Nse1-3-4 subcomplex cryo-EM samples. The microscopic examination has been repeated at least three samples independently with similar results. (c) Representative 2D classification of the Nse1-3-4 subcomplex. (d) The workflow of cryo-EM image processing and map reconstruction of Nse1-3-4 subcomplex. (e) Global Fourier Shell Correlation (FSC) curves (top) and Model vs. map FSC (bottom) of Nse1-3-4 subcomplex. (f) The angular distribution plot (right) and local resolution estimation of Nse1-3-4 cryo-EM map (left).

Source data

Extended Data Fig. 5 Cryo-EM structure determination of the Smc5/6-6mer.

(a) A SEC profile of the Smc5/6-6mer (left) with the peak fraction evaluated by SDS-PAGE (right). The sample purification has been repeated at least three times independently with similar results. (b) A representative micrograph of the Smc5/6-6mer. The microscopic examination has been repeated at least three samples independently with similar results. (c) Representative 2D classification of the Smc5/6-6mer. (d) The workflow of cryo-EM image processing and map reconstruction of Smc5/6-6mer.

Source data

Extended Data Fig. 6 Cryo-EM structure determination of the Smc5/6-5mer.

(a) A SEC profile of the Smc5/6-5mer (left) with the peak fraction evaluated by SDS-PAGE (right). The sample purification has been repeated at least three times independently with similar results. (b) A representative micrograph of the Smc5/6-5mer. The microscopic examination has been repeated at least three samples independently with similar results. (c) Representative 2D classification of the Smc5/6-5mer. (d) The workflow of cryo-EM image processing and map reconstruction.

Source data

Extended Data Fig. 7 A Nse3 loop involved in the binding of the Smc5 head region in the Smc5/6-6mer structure.

(a) Superimposition of the head regions of the Smc5/6-6mer structure obtained in this work and reported previously (PDB: 7QCD). (b) A Nse3153-172 loop with clear density map observed in the current Smc5/6-6mer structure (top and middle) was not resolved in the report structure (bottom; PDB: 7QCD). (c) The Nse3153-172 loop shows interaction with Nse1 and the Smc5 head region in the Smc5/6-6mer structure reported here (top). The residues involved in the interaction are in dark blue and are listed in the table (bottom). (d) The Nse3153-172 loop is seen in both the Smc5/6-6mer structure (color) and the Nse1-3-4 structure (gray) reported here. (e) The Nse3153-172 loop is near the ATP binding site of Smc6.

Extended Data Fig. 8 The elbow regions of three SMC complexes contain breaks at all four coiled coil helixes.

Cartoon representation of two SMC subunits in the budding yeast cohesin (PDB: 7OGT) (a), the budding yeast condensin (PDB: 6YVU) (b) and the bacteria MukBEF (PDB: 7NYY) (c). The elbow structures of these SMC complexes are marked with red boxes. A break at each of the four coiled-coil helix is seen (inset).

Extended Data Fig. 9 2D and 3D classification of Smc5/6 structures.

2D classification (left) or 3D classification (right) of Smc5/6-7mer (a) and Smc5/6-8mer structures (b) derived from negative staining EM data. These are partitioned into long and short particle groups. The percentage of long particle group is highlighted in color.

Extended Data Fig. 10 In vivo examination of Nse2-wedge and Nse6-hook mutations.

(a) Sequence alignment of Nse2 homologs from related yeast species at its wedge region. Saccharomyces species examined include S. cerevisiae (Scer), S. castellii (Scas), S. paradoxus (Spar), S. mikatae (Smik), and S. bayanus (Sbay). Identical and similar amino acids are indicated by black and gray circles, respectively, while non-conserved amino acids are not labeled. Three residues responsible for binding to the Smc6 arm are in red and were mutated in nse2-3A. (b) Quantification of the co-IP data to assess the effects of nse2-3A (left) and nse6-RAAE (right) on the association with Smc6 and Smc5, respectively. In each case, the mean (center line) from three trials (n = 3) using biologically independent samples (each dot representing one data) and standard deviation (limits) are shown. For statistical test, three biological independent samples over three independent experiments were examined. The means of the mutants are statistically different from those of the wild-type based on student t-test (two-sided) and not adjustment was made. For the co-IP data derived from nse2-3A vs. WT, the P values was 10−4 for both low or high salt buffer conditions. For the co-IP data derived from nse6-RAAR3 vs. WT, the P values was 0.0032 for low salt buffer conditions and 10−4 for high salt buffer conditions. (c) Sequence alignment of Nse6 homologs from related yeast species at its hook region focused on the front part. Saccharomyces species examined are annotated as in panel A. Four conserved residues (red, bold) involved in binding to Smc5-arm regions were mutated to generate nse6-RAAE, whereas the nse6-9A allele contains alanine substitution of D86-I94 (red). The short a1 helix (I88-I94) is marked. (d) Differential effects of nse6-RAAE and nse6-9A on Nse6 protein level as examined by immunoblotting using whole cell extract. At least two biological duplicates were examined over at least two independent experiments and only one representative result was shown.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–8 and Tables 1–3.

Reporting Summary

Supplementary Video 1

A video representing the structural changes of three Smc5/6 complexes and associated ATPase activities regulated by two subcomplexes, Nse1–3–4 or Nse5–6 in the opposite way.

Supplementary Video 2

A video representing Nse5–6 not hindering the head domain engagement in the conversion from Smc5/6-8mer to the reported structure (PDB: 7TVE) with docked Nse5–6.

Supplementary Data

Octet RED 96 Raw data of DNA binding activity results.

Source data

Source Data Fig. 1

Unprocessed gels.

Source Data Fig. 2

Statistical Source Data.

Source Data Fig. 3

Unprocessed western blots and unprocessed yeast culture plate.

Source Data Fig. 4

Unprocessed western blots and unprocessed yeast culture plate.

Source Data Extended Data Fig. 4

Unprocessed gels.

Source Data Extended Data Fig. 5

Unprocessed gels.

Source Data Extended Data Fig. 6

Unprocessed gels.

Source Data Extended Data Fig.10

Statistical Source Data.

Source Data Extended Data Fig.10

Unprocessed western blots.

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Li, Q., Zhang, J., Haluska, C. et al. Cryo-EM structures of Smc5/6 in multiple states reveal its assembly and functional mechanisms. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01319-1

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