Abstract
Bacteria have developed a variety of immune systems to combat phage infections. The Lamassu system is a prokaryotic immune system with a core conserved structural maintenance of chromosomes (SMC) superfamily protein LmuB and diverse effectors named LmuA, whose mechanism remains unclear. Here we present a series of cryo-electron microscopy structures of the type-I Lamassu complex from Bacillus cellulasensis and the type-II Lamassu complex from Vibrio cholerae, both in apo and dsDNA-bound states, revealing an unexpected stoichiometry and topological architecture distinct from canonical SMC complexes. Combined structural and biochemical analyses show how the nuclease effector LmuA is sequestered in an inactive monomeric form within the Lamassu complex and, upon sensing foreign DNA ends, dissociates and assembles into an active tetramer capable of DNA cleavage. Our findings elucidate the mechanism by which Lamassu systems detect viral replication and implement antiphage defense, highlighting the roles of SMC proteins in prokaryotic immunity.
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Data availability
Cryo-EM structures and atomic models generated in this study have been deposited in the wwPDB OneDep System under EMD accession codes EMD-64575, EMD-64576, EMD-64585, EMD-64586, EMD-64583 and EMD-64584 and PDB ID codes under accession codes 9UX7, 9UX8, 9UXK, 9UXL, 9UXH and 9UXI. Source data are provided with this paper.
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Acknowledgements
We thank the Cryo-EM Center at the University of Science and Technology of China (USTC) for their assistance with the cryo-EM experiments. This work is supported by National Key Research and Development Program of China (grant nos. 2024YFA0916903 to Y.Z., 2022YFC3401500 to Y.F., 2022YFA1302700 to K.Z., 2022YFC2303700 to K.Z. and 2022YFC2104800 to Y.F.), the National Natural Science Foundation of China (grant nos. 32371329 to Y.Z., 32171274 to Y.F., 32371345 to K.Z., 32301044 and 32471301 to S.L.), Scientific Research Innovation Capability Support Project for Young Faculty (grant no. ZYGXQNJSKYCXNLZCXM-B1) to Y.F., the Fundamental Research Funds for the Central Universities (grant no. QNTD2023-01) to Y.F., Anhui Provincial Natural Science Foundation (grant no. 2308085QC80) to S.L., the Strategic Priority Research Program of the Chinese Academy of Sciences (grant no. XDB0490000) to K.Z., the Center for Advanced Interdisciplinary Science and Biomedicine of IHM (grant no. QYPY20220019) to K.Z. and the USTC Research Funds of the Double First-Class Initiative (grant nos. YD9100002048 to K.Z. and YD9100002044 to S.L.).
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Y.Z., K.Z. and Y.F. conceived and supervised the project and designed experiments. M.L., Xingyu Z., D.L., W.X., Z.G., L.H. and L.A. purified the proteins and performed in vitro activity analysis and in vivo assays. M.L., Xiaolong Z., Y.G. and K.Z. collected the cryo-EM data and solved the cryo-EM structures. S.L. built and refined the models. Y.Z. wrote the original manuscript. Y.F., K.Z. and Y.Z. revised the manuscript.
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Extended data
Extended Data Fig. 1 Type I and type II Lamassu systems both form heterooligomers.
a–c, Purification profile of BcLmuA (a), BcLmuB (b) and BcLmuAB complex (c) on Superdex 200 column. d, SEC-MALS (Size Exclusion Chromatography with Multi-Angle Light Scattering) analysis of BcLmuB, BcLmuAB and VcLmuACB complex. Respective calculated molecular weight is marked on top of the peak. e, Statistics of the SEC-MALS results and theoretical molecular weight based on predicted complex ratios. f, g, Purification profile of VcLmuA (f) and VcLmuC (g) on Superdex-200 column. h, Purification test of His-SUMO-TEV-VcLmuB and SDS-PAGE gel of the samples. 1-bacteria pre-induced. 2-bacteria post-induced. 3-supernatant. 4-pellet. 5-flowthrough after Ni-column. 6-beads after wash. 7-Elution. 8-beads after elution. i, Purification profile of VcLmuACB complex on Superdex 200 column.
Extended Data Fig. 2 DNA binding, ATPase and nuclease activity of the Lamassu system.
a, EMSA to test the binding of BcLmuAK59AB and VcLmuAK57GCB to dsDNA. BcLmuAK59AB and VcLmuAK57GCB is included at a concentration of 0.25, 0.5, 1, 2, 4, 8 μM, respectively. b, pUC19 digestion by VcLmuACB in the presence or absence of ATP or ATPγS, respectively. c, Measurement of ATP or ATPγS hydrolysis activity for VcLmuACB and BcLmuAB at 0.5 μM final with or without 0.5 μM 59 bp DNA duplex, 0.25 mM ATP or ATPγS. Each group with three replicate measurements. Means and standard deviations are shown.
Extended Data Fig. 3 Single-particle cryo-EM analysis of the BcLmuAB complex.
a, Representative cryo-EM micrograph (upper) and reference-free 2D class averages (lower) of BcLmuAB. b, Workflow of the cryo-EM data processing. The final map resolution is color coded for different regions. c, Gold standard FSC plots for the 3D reconstructions of the whole map, calculated in cryoSPARC. d, Euler angle distribution of the particle images.
Extended Data Fig. 4 Single-particle cryo-EM data processing of VcLmuACB.
a, Representative cryo-EM micrograph of VcLmuACB. b, Representative cryoEM 2D averages of VcLmuACB. c, Cryo-EM data processing workflow for VcLmuACB. d, Gold standard FSC plot for the final 3D reconstruction of the whole map, calculated in cryoSPARC. e, Euler angle distribution of the particle images. f, The final map resolution is colored for different regions.
Extended Data Fig. 5 Single-particle cryo-EM data processing of VcLmuACB-pUC19.
a, Representative cryo-EM micrograph of VcLmuACB-pUC19. b, Reference-free 2D class averages of VcLmuACB-pUC19 c, Cryo-EM data processing workflow for VcLmuACB-pUC19. d, Gold standard FSC plot for the final 3D reconstruction. e, Euler angle distribution of the particle images. f, Resolution distribution (ResMap).
Extended Data Fig. 6 Structural alignment between different states of BcLmuAB and VcLmuACB complexes.
a, Structural alignment between the two apo state structures of VcLmuACB with different protein lengths. b–d, Structural alignment between the apo and DNA-bound states of BcLmuAB (b), between the apo (partial structure) and DNA-bound states of VcLmuACB (c), and between the apo (full length) and DNA-bound states of VcLmuACB (d).
Extended Data Fig. 7 Inter-subunit interactions in the apo state of VcLmuACB (full length).
a–e, Atomic model of the VcLmuACB complex is shown in a, in which detailed interactions between dsDNA and BcLmuAB are shown in b–e.
Extended Data Fig. 8 Single-particle cryo-EM analysis of the BcLmuAB-DNA complex.
a, Representative cryo-EM micrograph (left) and reference-free 2D class averages (right) of BcLmuAB-DNA. b, Workflow of the cryo-EM data processing. The final map resolution is color coded for different regions. c, Gold standard FSC plots for the 3D reconstructions of the whole map, calculated in cryoSPARC (left) and Euler angle distribution of the particle image(right).
Extended Data Fig. 9 Single-particle cryo-EM data processing of VcLmuACB-59bp DNA complex and VcLmuA-tetramer complex.
a, Representative cryo-EM micrograph of VcLmuACB-59bp DNA. b, Workflow of cryo-EM data processing of VcLmuACB-59bp DNA. c, Reference-free 2D class average of VcLmuACB-DNA. d, Reference-free 2D class average of VcLmuA-tetramer. e, Gold standard FSC plots for the 3D reconstructions of the whole map of VcLmuACB-DNA, calculated in cryoSPARC. f, Gold standard FSC plots for the 3D reconstructions of the whole map of VcLmuA-tetramer, calculated in cryoSPARC. g, Euler angle distribution of the particle image of VcLmuACB-DNA. h, Euler angle distribution of the particle image of VcLmuA-tetramer. i, The final map resolution is color coded for different regions of VcLmuACB-DNA. j, The final map resolution is color coded for different regions of VcLmuA-tetramer.
Extended Data Fig. 10 Enzymatic cleavage activity towards five types of DNA substrates.
a, Schematic representation and sequences of the five DNA structures. b–f, Time-course cleavage of double-stranded (b), single-stranded (c), palindromic DNA (d), circular DNA (pUC19) (e) and Linear phage DNA (extracted from phage T1) (f) by two Lamassu systems. The reaction contains 80 nM VcLmuACB or 20 nM BcLmuAB and 1 μM DNA substrates (b–d), 22 nM pUC19 or 1.2 nM T1 phage genomic DNA.
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Li, M., Zhao, X., Zhao, X. et al. Structural insights into type-I and type-II Lamassu antiphage systems. Nat Chem Biol (2026). https://doi.org/10.1038/s41589-025-02102-z
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DOI: https://doi.org/10.1038/s41589-025-02102-z
