Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Cryo-EM structures of LHCII in photo-active and photo-protecting states reveal allosteric regulation of light harvesting and excess energy dissipation

Nature Plants volume 9, pages 1547–1557 (2023)Cite this article

Subjects

Abstract

The major light-harvesting complex of photosystem II (LHCII) has a dual regulatory function in a process called non-photochemical quenching to avoid the formation of reactive oxygen. LHCII undergoes reversible conformation transitions to switch between a light-harvesting state for excited-state energy transfer and an energy-quenching state for dissipating excess energy under full sunshine. Here we report cryo-electron microscopy structures of LHCII in membrane nanodiscs, which mimic in vivo LHCII, and in detergent solution at pH 7.8 and 5.4, respectively. We found that, under low pH conditions, the salt bridges at the lumenal side of LHCII are broken, accompanied by the formation of two local α-helices on the lumen side. The formation of α-helices in turn triggers allosterically global protein conformational change, resulting in a smaller crossing angle between transmembrane helices. The fluorescence decay rates corresponding to different conformational states follow the Dexter energy transfer mechanism with a characteristic transition distance of 5.6 Å between Lut1 and Chl612. The experimental observations are consistent with the computed electronic coupling strengths using multistate density function theory.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Cryo-EM structures of LHCII in nanodisc and in detergent solution.
Fig. 2: Protein secondary structures and pigments in different conformations.
Fig. 3: Relationships of key structural factors related to state transitions.
Fig. 4: Key structural factors that drive state transition.
Fig. 5: Fluorescence spectra indicate the fluorescence reversibility.

Similar content being viewed by others

Data availability

The cryo-EM maps of spinach LHCII in nanodisc and in detergent solution at pH 7.8 or pH 5.4 have been deposited in the Electron Microscopy Data Bank under the accession codes EMD-35785, EMD-35786, EMD-35787, EMD-35782, EMD-35783 and EMD-35784. The corresponding structure models are deposited in the Protein Data Bank (PDB) under accession codes 8IX0, 8IX1, 8IX2, 8IWX, 8IWY and 8IWZ. The LHCII crystal structures used in this article can be accessed in the PDB using the accession codes 1RWT and 2BHW.

References

  1. Nicol, L., Nawrocki, W. J. & Croce, R. Disentangling the sites of non-photochemical quenching in vascular plants. Nat. Plants 5, 1177–1183 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Demmig-Adams, B., Garab, G., Adams, W. III & Govindjee (eds) Non-Photochemical Quenching and Energy Dissipation in Plants, Algae and Cyanobacteria (Springer, 2014).

  3. Horton, P., Ruban, A. V. & Walters, R. G. Regulation of light harvesting in green algae. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 655–684 (1996).

    Article  CAS  PubMed  Google Scholar 

  4. Ruban, A. V. Light harvesting control in plants. FEBS Lett. 592, 3030–3039 (2018).

    Article  CAS  PubMed  Google Scholar 

  5. Li, X. P. et al. Regulation of photosynthetic light harvesting involves intrathylakoid lumen pH sensing by the PsbS protein. J. Biol. Chem. 279, 22866–22874 (2004).

    Article  CAS  PubMed  Google Scholar 

  6. Ruban, A. V. & Wilson, S. The mechanism of non-photochemical quenching in plants: localization and driving forces. Plant Cell Physiol. 62, 1063–1072 (2021).

    Article  CAS  PubMed  Google Scholar 

  7. Murchie, E. H. & Niyogi, K. K. Manipulation of photoprotection to improve plant photosynthesis. Plant Physiol. 155, 86–92 (2011).

    Article  CAS  PubMed  Google Scholar 

  8. Dall’Osto, L. et al. Two mechanisms for dissipation of excess light in monomeric and trimeric light-harvesting complexes. Nat. Plants 3, 17033 (2017).

    Article  PubMed  Google Scholar 

  9. De Souza, A. P. et al. Soybean photosynthesis and crop yield are improved by accelerating recovery from photoprotection. Science 377, 851–854 (2022).

    Article  PubMed  Google Scholar 

  10. Kromdijk, J. et al. Improving photosynthesis and crop productivity by accelerating recovery from photoprotection. Science 354, 857–861 (2016).

    Article  CAS  PubMed  Google Scholar 

  11. Liu, Z. et al. Crystal structure of spinach major light-harvesting complex at 2.72 Å resolution. Nature 428, 287–292 (2004).

    Article  CAS  PubMed  Google Scholar 

  12. Huyer, J. et al. Fluorescence decay kinetics of solubilized pigment protein complexes from the distal, proximal, and core antenna of photosystem II in the range of 10-277 K and absence or presence of sucrose. J. Phys. Chem. B 108, 3326–3334 (2004).

    Article  CAS  Google Scholar 

  13. Palacios, M. A., de Weerd, F. L., Ihalainen, J. A., van Grondelle, R. & van Amerongen, H. Superradiance and exciton (de) localization in light-harvesting complex II from green plants? J. Phys. Chem. B 106, 5782–5787 (2002).

    Article  CAS  Google Scholar 

  14. Moya, I., Silvestri, M., Vallon, O., Cinque, G. & Bassi, R. Time-resolved fluorescence analysis of the photosystem II antenna proteins in detergent micelles and liposomes. Biochemistry 40, 12552–12561 (2001).

    Article  CAS  PubMed  Google Scholar 

  15. van Oort, B., van Hoek, A., Ruban, A. V. & van Amerongen, H. Aggregation of light-harvesting complex II leads to formation of efficient excitation energy traps in monomeric and trimeric complexes. FEBS Lett. 581, 3528–3532 (2007).

    Article  PubMed  Google Scholar 

  16. Vasil’ev, S. et al. Quenching of chlorophyll a fluorescence in the aggregates of LHCII: steady state fluorescence and picosecond relaxation kinetics. Biochemistry 36, 7503–7512 (1997).

    Article  PubMed  Google Scholar 

  17. Liguori, N., Periole, X., Marrink, S. J. & Croce, R. From light-harvesting to photoprotection: structural basis of the dynamic switch of the major antenna complex of plants (LHCII). Sci. Rep. 5, 15661 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Van Oort, B. et al. Different crystal morphologies lead to slightly different conformations of light-harvesting complex II as monitored by variations of the intrinsic fluorescence lifetime. Phys. Chem. Chem. Phys. 13, 12614–12622 (2011).

    Article  PubMed  Google Scholar 

  19. Barros, T., Royant, A., Standfuss, J., Dreuw, A. & Kuhlbrandt, W. Crystal structure of plant light-harvesting complex shows the active, energy-transmitting state. EMBO J. 28, 298–306 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Horton, P. et al. Control of the light-harvesting function of chloroplast membranes by aggregation of the LHCII chlorophyll–protein complex. FEBS Lett. 292, 1–4 (1991).

    Article  CAS  PubMed  Google Scholar 

  21. Tutkus, M., Chmeliov, J., Rutkauskas, D., Ruban, A. V. & Valkunas, L. Influence of the carotenoid composition on the conformational dynamics of photosynthetic light-harvesting complexes. J. Phys. Chem. Lett. 8, 5898–5906 (2017).

    Article  CAS  PubMed  Google Scholar 

  22. Schlau-Cohen, G. S. et al. Single-molecule identification of quenched and unquenched states of LHCII. J. Phys. Chem. Lett. 6, 860–867 (2015).

    Article  CAS  PubMed  Google Scholar 

  23. Yan, H., Zhang, P., Wang, C., Liu, Z. & Chang, W. Two lutein molecules in LHCII have different conformations and functions: insights into the molecular mechanism of thermal dissipation in plants. Biochem. Biophys. Res. Commun. 355, 457–463 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. Ruban, A. V. et al. Identification of a mechanism of photoprotective energy dissipation in higher plants. Nature 450, 575–578 (2007).

    Article  CAS  PubMed  Google Scholar 

  25. Standfuss, J., Terwisscha van Scheltinga, A. C. T., Lamborghini, M. & Kühlbrandt, W. Mechanisms of photoprotection and nonphotochemical quenching in pea light-harvesting complex at 2.5 A resolution. EMBO J. 24, 919–928 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Pascal, A. A. et al. Molecular basis of photoprotection and control of photosynthetic light-harvesting. Nature 436, 134–137 (2005).

    Article  CAS  PubMed  Google Scholar 

  27. Daskalakis, V. et al. Structural basis for allosteric regulation in the major antenna trimer of photosystem II. J. Phys. Chem. B 123, 9609–9615 (2019).

    Article  CAS  PubMed  Google Scholar 

  28. Li, H. et al. Dynamical and allosteric regulation of photoprotection in light harvesting complex II. Sci. China Chem. 63, 1121–1133 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Daskalakis, V., Papadatos, S. & Stergiannakos, T. The conformational phase space of the photoprotective switch in the major light harvesting complex II. Chem. Commun. 56, 11215–11218 (2020).

    Article  CAS  Google Scholar 

  30. Navakoudis, E., Stergiannakos, T. & Daskalakis, V. A perspective on the major light-harvesting complex dynamics under the effect of pH, salts, and the photoprotective PsbS protein. Photosynth. Res. 156, 163–177 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Ruban, A. V., Johnson, M. P. & Duffy, C. D. The photoprotective molecular switch in the photosystem II antenna. Biochim. Biophys. Acta 1817, 167–181 (2012).

    Article  CAS  PubMed  Google Scholar 

  32. Son, M., Pinnola, A., Gordon, S. C., Bassi, R. & Schlau-Cohen, G. S. Observation of dissipative chlorophyll-to-carotenoid energy transfer in light-harvesting complex II in membrane nanodiscs. Nat. Commun. 11, 1295 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Mascoli, V. et al. Capturing the quenching mechanism of light-harvesting complexes of plants by zooming in on the ensemble. Chemistry 5, 2900–2912 (2019).

    Article  CAS  Google Scholar 

  34. Maity, S., Daskalakis, V., Elstner, M. & Kleinekathofer, U. Multiscale QM/MM molecular dynamics simulations of the trimeric major light-harvesting complex II. Phys. Chem. Chem. Phys. 23, 7407–7417 (2021).

    Article  CAS  PubMed  Google Scholar 

  35. Madjet, M. E.-A., Müh, F. & Renger, T. Deciphering the influence of short-range electronic couplings on optical properties of molecular dimers: application to ‘special pairs’ in photosynthesis. J. Phys. Chem. B 113, 12603–12614 (2009).

    Article  CAS  PubMed  Google Scholar 

  36. Saccon, F. et al. A protein environment-modulated energy dissipation channel in LHCII antenna complex. iScience 23, 101430 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ilioaia, C., Johnson, M. P., Horton, P. & Ruban, A. V. Induction of efficient energy dissipation in the isolated light-harvesting complex of photosystem II in the absence of protein aggregation. J. Biol. Chem. 283, 29505–29512 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Yamano, N., Wang, P., Dong, F. Q. & Zhang, J. P. Lipid-enhanced photoprotection of LHCII in membrane nanodisc by reducing chlorophyll triplet production. J. Phys. Chem. B 126, 2669–2676 (2022).

    Article  CAS  PubMed  Google Scholar 

  39. Manna, P. & Schlau-Cohen, G. S. Photoprotective conformational dynamics of photo synthetic light-harvesting proteins. Biochim. Biophys. Acta Bioenerg. 1863, 148543 (2022).

    Article  CAS  PubMed  Google Scholar 

  40. Pandit, A. et al. Assembly of the major light-harvesting complex II in lipid nanodiscs. Biophys. J. 101, 2507–2515 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Tietz, S. et al. A proteoliposome-based system reveals how lipids control photosynthetic light harvesting. J. Biol. Chem. 295, 1857–1866 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Van Den Brink-Van Der Laan, E., Antoinette Killian, J. & De Kruijffu, B. Nonbilayer lipids affect peripheral and integral membrane proteins via changes in the lateral pressure profile. Biochim. Biophys. Acta Biomembr. 1666, 275–288 (2004).

    Article  Google Scholar 

  43. Manna, P., Davies, T., Hoffmann, M., Johnson, M. P. & Schlau-Cohen, G. S. Membrane-dependent heterogeneity of LHCII characterized using single-molecule spectroscopy. Biophys. J. 120, 3091–3102 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Johansen, N. T. et al. Structural and biophysical properties of supercharged and circularized nanodiscs. Langmuir 37, 6681–6690 (2021).

    Article  CAS  PubMed  Google Scholar 

  45. Goral, T. K. et al. Light-harvesting antenna composition controls the macrostructure and dynamics of thylakoid membranes in Arabidopsis. Plant J. 69, 289–301 (2012).

    Article  CAS  PubMed  Google Scholar 

  46. Nicol, L. & Croce, R. The PsbS protein and low pH are necessary and sufficient to induce quenching in the light-harvesting complex of plants LHCII. Sci. Rep. 11, 7415 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Betterle, N. et al. Light-induced dissociation of an antenna hetero-oligomer is needed for non-photochemical quenching induction. J. Biol. Chem. 284, 15255–15266 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Johnson, M. P. & Ruban, A. V. Restoration of rapidly reversible photoprotective energy dissipation in the absence of PsbS protein by enhanced ΔpH. J. Biol. Chem. 286, 19973–19981 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Horton, P., Wentworth, M. & Ruban, A. V. Control of the light harvesting function of chloroplast membranes: the LHCII-aggregation model for non-photochemical quenching. FEBS Lett. 579, 4201–4206 (2005).

    Article  CAS  PubMed  Google Scholar 

  50. Azadi-Chegeni, F. et al. Protein dynamics and lipid affinity of monomeric, zeaxanthin-binding LHCII in thylakoid membranes. Biophys. J. 121, 396–409 (2022).

    Article  CAS  PubMed  Google Scholar 

  51. Wentworth, M., Ruban, A. V. & Horton, P. Thermodynamic investigation into the mechanism of the chlorophyll fluorescence quenching in isolated photosystem II light-harvesting complexes. J. Biol. Chem. 278, 21845–21850 (2003).

    Article  CAS  PubMed  Google Scholar 

  52. Tang, Y. et al. Heat stress induces aggregation of the light harvesting complex of photosystem II in spinach plants. Plant Physiol. 143, 629–638 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Janik, E. et al. Molecular architecture of plant thylakoids under physiological and light stress conditions: a study of lipid-light-harvesting complex II model membranes. Plant Cell 25, 2155–2170 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Havaux, M. Carotenoids as membrane stabilizers in chloroplasts. Trends Plant Sci. 3, 147–151 (1998).

    Article  Google Scholar 

  55. Tardy, F. & Havaux, M. Thylakoid membrane fluidity and thermostability during the operation of the xanthophyll cycle in higher-plant chloroplasts. Biochim. Biophys. Acta 1330, 179–193 (1997).

    Article  CAS  PubMed  Google Scholar 

  56. Gruszecki, W. I. & Sielewiesiuk, J. Galactolipid multibilayers modified with xanthophylls: orientational and diffractometric studies. Biochim. Biophys. Acta 1069, 21–26 (1991).

    Article  CAS  PubMed  Google Scholar 

  57. Seiwert, D., Witt, H., Janshoff, A. & Paulsen, H. The non-bilayer lipid MGDG stabilizes the major light-harvesting complex (LHCII) against unfolding. Sci. Rep. 7, 5158 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  58. Rutkauskas, D., Chmeliov, J., Johnson, M., Ruban, A. & Valkunas, L. Exciton annihilation as a probe of the light-harvesting antenna transition into the photoprotective mode. Chem. Phys. 404, 123–128 (2012).

    Article  CAS  Google Scholar 

  59. Caffarri, S., Kouril, R., Kereiche, S., Boekema, E. J. & Croce, R. Functional architecture of higher plant photosystem II supercomplexes. EMBO J. 28, 3052–3063 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Ritchie, T. et al. Reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods Enzymol. 464, 211–231 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Wu, C. L., Huang, X. J., Cheng, J., Zhu, D. J. & Zhang, X. Z. High-quality, high-throughput cryo-electron microscopy data collection via beam tilt and astigmatism-free beam-image shift. J. Struct. Biol. 208, 107396 (2019).

    Article  CAS  PubMed  Google Scholar 

  62. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  PubMed  Google Scholar 

  63. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  64. Pettersen, E. F. et al. UCSF chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  65. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  67. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).

    Article  CAS  PubMed  Google Scholar 

  68. Robert, X. & Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–W324 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Grofe, A. et al. Generalization of block-localized wave function for constrained optimization of excited determinants. J. Chem. Theory Comput. 17, 277–289 (2021).

    Article  CAS  PubMed  Google Scholar 

  70. Sirohiwal, A., Berraud-Pache, R., Neese, F., Izsak, R. & Pantazis, D. A. Accurate computation of the absorption spectrum of chlorophyll alpha with pair natural orbital coupled cluster methods. J. Phys. Chem. B 124, 8761–8771 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Dreuw, A. Influence of geometry relaxation on the energies of the S1 and S2 states of violaxanthin, zeaxanthin, and lutein. J. Phys. Chem. A 110, 4592–4599 (2006).

    Article  CAS  PubMed  Google Scholar 

  72. Zhao, R., Hettich, C. P. & Chen, X. Minimal-active-space multistate density functional theory for excitation energy involving local and charge transfer states. npj Comput. Mater. 7, 148 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Zhao, R. et al. Dynamic-then-static approach for core excitations of open-shell molecules. J. Phys. Chem. Lett. 12, 7409–7417 (2021).

    Article  CAS  PubMed  Google Scholar 

  74. Gao, J., Grofe, A., Ren, H. & Bao, P. Beyond Kohn–Sham approximation: hybrid multistate wave function and density functional theory. J. Phys. Chem. Lett. 7, 5143–5149 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Science and Beijing Branch of Songshan Lake Laboratory for Materials Science for our cryo-EM work. We thank the Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Science for our cryo-EM work, and we thank B. Zhu, X. Huang and L. Chen for their help taking EM images. We thank the cryo-EM centre of the Southern University of Science and Technology for our cryo-EM work and we thank L. Fu, J. Wu and S. Xu for their help taking EM images. We thank T. Kuang for encouragement and M. Li for in-depth discussion. We thank H. Yan for sending us the crystal structure data of LHCII from cucumber. This work was supported by the Chinese Academy of Sciences (grant nos. QYZDJ-SSW-SYS017, XDB33000000 and YJKYYQ20170046 to Y. Weng), the National Natural Science Foundation of China (grant no. 11721404 to Y. Weng) and the Shenzhen Municipal Science and Technology Innovation Commission (grant no. KQTD2017-0330155106581 to J.G.).

Author information

Author notes

  1. These authors contributed equally: Meixia Ruan, Hao Li.

Authors and Affiliations

  1. Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China

    Meixia Ruan, Ying Zhang, Zhuan Wang, Yumei Wang, Dapeng Sun, Wei Ding & Yuxiang Weng

  2. University of Chinese Academy of Sciences, Beijing, China

    Meixia Ruan, Ying Zhang, Wei Ding & Yuxiang Weng

  3. School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China

    Hao Li & Jiali Gao

  4. Institute of Systems and Physical Biology, Shenzhen Bay Laboratory, Shenzhen, China

    Hao Li, Ruoqi Zhao, Jun Zhang, Yingjie Wang & Jiali Gao

  5. Department of Chemistry and Supercomputing Institute, University of Minnesota, Minneapolis, MN, USA

    Jiali Gao

  6. Songshan Lake Materials Laboratory, Dongguan, China

    Yuxiang Weng

Authors
  1. Meixia Ruan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hao Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ying Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ruoqi Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jun Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yingjie Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jiali Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Zhuan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yumei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Dapeng Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Wei Ding
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Yuxiang Weng
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.R. and H.L. purified samples and collected cryo-EM data. Y.Z., Z.W., W.D., Yumei Wang and D.S. assisted with data collection. W.D. processed cryo-EM data and reconstructed the density map. M.R., H.L. and Y. Weng analysed the structures. R.Z. and J.Z. wrote the software. R.Z., Yingjie Wang and J.G. calculated and analysed the electronic coupling. H.L. characterized and analysed the fluorescence spectra and lifetime measurement. The article was written by M.R., W.D., J.G. and Y. Weng with contributions by all authors. M.R., H.L., W.D. and Y. Weng prepared all figures. Y. Weng conceived of and coordinated the whole project.

Corresponding authors

Correspondence to Jiali Gao, Wei Ding or Yuxiang Weng.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Plants thanks Mei Li and Nicoletta Liguor for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Sample purification of LHCII and LHCII nanodisc.

a, Sucrose density gradient ultra-centrifugation separation of LHCII trimer. b, SDS-PAGE of LHCII nanodisc, LHCII in detergent solution and membrane scaffold protein MSP1E3D1. The experiment was repeated three times independently with similar results. c, Absorption trace at 280 nm and 672 nm during the Ni-NTA column purification of LHCII nanodisc. d, Absorption trace at 280 nm and 672 nm during size exclusion chromatography column purification of LHCII nanodisc.

Extended Data Fig. 2 Fluorescence decay kinetics and UV-vis and FTIR absorption spectra of LHCII nanodisc and LHCII in detergent solution.

All spectra are the averaged results of three measurements. a, Fluorescence decay kinetics of LHCII in 0.03% β-DDM at pH 7.8 and 5.4 and LHCII nanodisc at pH 7.8 and 5.4 respectively, excited at 480 nm laser with a repetition frequency of 100 kHz, an average power density of 1.5 mW/cm2, an instrumental response factor (IRF) of 0.115 ns. b, UV-vis absorption spectra of LHCII in detergent solution and LHCII nanodisc. c, Secondary derivative FTIR spectra of LHCII trimer in 0.03% DDM at pH 7.8 and 5.4 respectively. d, Secondary derivative FTIR spectra of LHCII nanodisc at pH 7.8 and 5.4 respectively. e, Lifetime constants and the associated amplitudes of LHCII in different environments based on biexponential fitting.

Extended Data Fig. 3 Structural analysis flow chart of LHCII nanodisc at pH 7.8 (a) and 5.4 (b).

a, I, A representative cryo-EM image of 8,894 collected for LHCII nanodisc at pH 7.8. II, 2D class averages of characteristic projection views of cryo-EM particles selected for further processing. III, Gold-standard Fourier Shell Correlation (FSC) curves of unprotonated conformation at pH 7.8, the 0.143 cut-off value is indicated by a horizontal blue line. IV, Flowchart for cryo-EM data processing. V, Angular distribution plot of particles used for final 3D refinement. The distribution was calculated with CryoSPARC 4.0. The different colors indicate the different number of particles that have such orientations according to the bar shown on the right. VI, Local resolution map analyzed by the local resolution estimation tool in CryoSPARC. b, Protonated (left) and unprotonated (right) conformation at pH 5.4; the detailed illustrations of I, II, III, IV, V and VI are the same as those in a.

Extended Data Fig. 4 Structural analysis flow chart of LHCII in detergent solution at pH 7.8 (a) and 5.4 (b).

a, I, A representative cryo-EM image of 7,282 collected for LHCII in detergent solution at pH 7.8. II, 2D class averages of characteristic projection views of cryo-EM particles selected for further processing. III, Gold-standard Fourier Shell Correlation (FSC) curves of unprotonated conformation at pH 7.8, the 0.143 cut-off value is indicated by a horizontal blue line. IV, Flowchart for cryo-EM data processing. V, Angular distribution plot of particles used for final 3D refinement. The distribution was calculated with CryoSPARC 4.0. The different colors indicate the different number of particles that have such orientations according to the bar shown on the right. VI, Local resolution map analyzed by the local resolution estimation tool in CryoSPARC. b, Protonated (left) and unprotonated (right) conformation at pH 5.4; the detailed illustrations of I, II, III, IV, V and VI are the same as those in a.

Extended Data Fig. 5 Comparison of protein secondary structures and pigments in different conformations.

a, b, Formation or disruption of salt bridge between K203 and E207 (a) at lumenal side and hydrogen bonds network among D54 (b) at stromal side of each monomer in the unprotonated (green) and protonated (magenta) conformations of LHCII in detergent solution, suggesting the protonation of E207 and D54 in LHCII after acidification. c, Average distance for K203-E207 and D54-D54 in different conformations. &: D54-D54 between three monomers. #: Unprotonated conformation at low pH (5.4) condition. &: Protonated conformation at low pH (5.4) condition. Data in bracket are the standard deviations of the average values. d, T57 and N61 in unprotonated (pink) and protonated (teal) conformations for LHCII nanodisc, the black arrow indicates the conformational transitions associated with protonation. e, Alignment of unprotonated structures at pH 7.8 (pink, green) and pH 5.4 (light blue, yellow) of LHCII in nanodisc (left) and in detergent solution (right). f, g, Structural comparison for helix E (f) and C-terminal (g) of LHCII in detergent solution without (pH 7.8, left) and with acidification (pH 5.4, right), a change from 310-helix or C-terminal random coil to α-helix is observed, along with C-terminal retraction towards helix D. h, Nex alignment of unprotonated structure at pH 7.8 (pink; green) and corresponding protonated structure at pH 5.4 (teal; magenta) of LHCII nanodisc (left) and LHCII in detergent solution (right), respectively, and a twist of the hexyl ring at stromal side occurs upon acidification for LHCII in nanodisc (expanded view). i, Lut1 and adjacent Chl610 pigment alignments of unprotonated structure at pH 7.8 (pink) and corresponding protonated structure at pH 5.4 (teal) of LHCII nanodisc, Lut1-Chl610 distance is 6.15 Å and 5.58 Å respectively, characterized by the Mg atom of Chl610 and the C27 atom in the conjugated π-system of Lut1. j, Vio alignment of unprotonated structure at pH 7.8 (pink; green) and corresponding protonated structure at pH 5.4 (teal; magenta) of LHCII in nanodisc (left) and in detergent solution (right).

Extended Data Fig. 6 Electron-density map and resolution of local structures and pigments of unprotonated structures at pH 7.8 and protonated structures at pH 5.4 for LHCII in nanodisc and in detergent solution respectively.

Local structure of pigments is double checked in COOT with best real space refinement statistics, such as Bonds, Angles, Torsions, Planes, Chirals, Non-bonded and Rama Plot. a, Local structural density map that involved D54-D54 and K203-E207 for LHCII in nanodisc (upper panel) and in detergent solution (lower panel), unprotonated structures are to the left of the dashed line (the key residues are shown in green (pH 7.8) or yellow (pH 5.4)) and protonated structures are at right (key residues are shown in blue). b, Density map of local structures and pigments for the unprotonated conformation at pH 7.8 (left) and protonated conformation at pH 5.4 (right) of LHCII in nanodisc. c, Density map of local structures and pigments for the unprotonated conformation at pH 7.8 (left) and protonated conformation at pH 5.4 (right) of LHCII in detergent solution. d, Local resolution and local correlation coefficients (in bracket, model vs map) for significant structures in different LHCII conformations, analyzed by phenix.validation_cryoem. #: Protonated conformation at pH 5.4.

Extended Data Fig. 7 Structural factors related to state transition at different conditions and their relationships.

a, Plot of Lut1-Chl610 electronic coupling strength \({\left.\ \right|V}_{{Q}_{y}^{{Chl}610},{S}_{1}^{{Lut}1}}{\left.\ \right|}^{2}/10000\) against Lut1-Chl610 separation distance in different LHCII structures. b, Plots of the fluorescence decay rate (k = 1/fluorescence lifetime, black solid circles), the summed coupling strength\({\left.\ \right|V}_{{Q}_{y}^{{Chl}612},{S}_{1}^{{Lut}1}}+{V}_{{Q}_{y}^{{Chl}610},{S}_{1}^{{Lut}1}}{\left.\ \right|}^{2}/10000\) (purple solid circles) of Lut1–Chl612 and Lut1-Chl610 pairs against the Lut1-Chl612 separation distance (R), and the fitting equation is \({k}=0.31+0.31{{\rm{e}}}^{-25\left({\rm{R}}-5.6\right)}\). c, Plot of available fluorescence lifetime (black star represents the data from the current work, blue solid circles and triangles represent data from the literatures38,39,40) and flexibility (orange solid circles, data from the literature44) of LHCII in nanodisc against the corresponding nanodisc size. d, Plot of helix D-E distance against Lut1-Chl612 separation distance from different LHCII structures, red dotted line marks the critical separation distance of 5.6 Å, green solid circles represent the data from the crystal structures (PDB code: 1RWT, 2BHW).

Extended Data Fig. 8 Configurations and excitation energies for the first singlet excited states of the chlorophyll monomer and lutein monomer.

a, Depiction of the minimal number of configurations necessary to model first singlet excited states of the chlorophyll monomer and lutein monomer. The ground-state configuration (Ψ0) is shown along with eight spin-contaminated configurations (1–8). b, Excitation energies of chlorophyll and lutein and the reference values.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics of LHCII structures at different conditions
Full size table
Extended Data Table 2 Structural parameters for LHCII at different conditions. &: Protonated structures at pH 5.4. *: Data not available. #: Salt bridge separation distance, which are slightly affected by acidification. Data in bracket are the standard deviations of the average values
Full size table

Supplementary information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ruan, M., Li, H., Zhang, Y. et al. Cryo-EM structures of LHCII in photo-active and photo-protecting states reveal allosteric regulation of light harvesting and excess energy dissipation. Nat. Plants 9, 1547–1557 (2023). https://doi.org/10.1038/s41477-023-01500-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41477-023-01500-2

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing