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Structural insights into a unique PSI–LHCI–LHCII–Lhcb9 supercomplex from moss Physcomitrium patens

Nature Plants volume 9, pages 832–846 (2023)Cite this article

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Abstract

Photosystem I (PSI) possesses a variable supramolecular organization among different photosynthetic organisms to adapt to different light environments. Mosses are evolutionary intermediates that diverged from aquatic green algae and evolved into land plants. The moss Physcomitrium patens (P. patens) has a light-harvesting complex (LHC) superfamily more diverse than those of green algae and higher plants. Here, we solved the structure of a PSI–LHCI–LHCII–Lhcb9 supercomplex from P. patens at 2.68 Å resolution using cryo-electron microscopy. This supercomplex contains one PSI–LHCI, one phosphorylated LHCII trimer, one moss-specific LHC protein, Lhcb9, and one additional LHCI belt with four Lhca subunits. The complete structure of PsaO was observed in the PSI core. One Lhcbm2 in the LHCII trimer interacts with PSI core through its phosphorylated N terminus, and Lhcb9 mediates assembly of the whole supercomplex. The complicated pigment arrangement provided important information for possible energy-transfer pathways from the peripheral antennae to the PSI core.

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Fig. 1: Sample preparation and characterization of the PSI–LHCI–LHCII–Lhcb9 supercomplex from P. patens.
Fig. 2: Overall architecture of the Pp PSI–LHCI–LHCII–Lhcb9 supercomplex.
Fig. 3: Comparison of the Pp PSI–LHCI–LHCII–Lhcb9 structure with those of maize PSI–LHCI–LHCII and a green algal PSI–LHCI–LHCII.
Fig. 4: Structural features of individual PSI core and antenna subunits in Pp PSI–LHCI–LHCII–Lhcb9.
Fig. 5: Structure of the phosphorylation site of Lhcbm2c and its interactions with the PSI core subunits in P. patens.
Fig. 6: Interactions between Lhcb9 and its adjacent subunits.
Fig. 7: Interactions between the subunits from PSI–LHCI–LHCII and outer layer of LHCI belt II in Pp PSI–LHCI–LHCII–Lhcb9.
Fig. 8: Possible EET pathways from peripheral antenna complexes to the reaction centre in Pp PSI–LHCI–LHCII–Lhcb9.

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Data availability

The cryo-EM density map and atomic model for the PSI–LHCI–LHCII–Lhcb9 supercomplex structure at 2.68 Å resolution have been deposited in the Electron Microscopy Data Bank and the Protein Data Bank (EMD ID code 33401 and PDB ID code 7XQP). The data that support the findings of this study are available from the corresponding authors upon request. Source data are provided with this paper.

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Acknowledgements

We thank W. Tang and Y. Yin from the Institute of Botany, Chinese Academy of Sciences (CAS) for instrumental supports in sample preparation, fluorescence measurement and HPLC analysis; the Centre of Cryo-Electron Microscopy, Zhejiang University for their technical assistance on cryo-EM data collection; C. Ma from the Protein Facility, School of Medicine, Zhejiang University for providing the platform for sample purification; and X. Meng at the Centre of Biomedical Analysis, Tsinghua University for protein MS analysis. The project was funded by the National Key R&D Programme of China (2022YFC1803400, 2022YFA0911900, 2020YFA0907600, 2018YFA0507700, 2019YFA0906300, 2021YFA1300403), CAS Project for Young Scientists in Basic Research (YSBR-004), the Strategic Priority Research Programme of CAS (XDA27050402), CAS Interdisciplinary Innovation Team (JCTD-2020-06), Youth Innovation Promotion Association of CAS (2020081), the Fundamental Research Funds for the Central Universities (2018XZZX001-13) and KAKENHI JP22H04916.

Author information

Author notes

  1. These authors contributed equally: Song Zhang, Kailu Tang, Qiujing Yan.

Authors and Affiliations

  1. Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China

    Song Zhang, Qiujing Yan, Xingyue Li, Liangliang Shen, Wenda Wang, Tingyun Kuang, Guangye Han & Jian-Ren Shen

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

    Song Zhang, Qiujing Yan, Xingyue Li & Liangliang Shen

  3. Department of Biophysics and Department of Pathology of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China

    Kailu Tang & Xing Zhang

  4. China National Botanical Garden, Beijing, China

    Wenda Wang, Tingyun Kuang, Guangye Han & Jian-Ren Shen

  5. College of Life Sciences, Capital Normal University, Beijing, China

    Yi-Kun He

  6. The Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing, China

    Jian-Ren Shen

  7. Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan

    Jian-Ren Shen

  8. Center of Cryo-Electron Microscopy, Zhejiang University School of Medicine, Hangzhou, China

    Xing Zhang

  9. Liangzhu Laboratory, Zhejiang University Medical Center, Hangzhou, China

    Xing Zhang

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Contributions

G.H., J.-R.S. and X.Z. conceived the project. S.Z., Q.Y. and G.H. performed the sample preparation and characterization. K.T. collected the cryo-EM data. X.Z. processed the cryo-EM data and reconstructed the cryo-EM map. S.Z., X.L., L.S. and W.W. built the structural model and refined the structure. S.Z., G.H., X.Z. and J.-R.S. analysed the structure. S.Z., K.T., G.H., X.Z. and J.-R.S. jointly wrote the manuscript. All authors discussed and commented on the results and the manuscript.

Corresponding authors

Correspondence to Guangye Han, Jian-Ren Shen or Xing Zhang.

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Nature Plants thanks Roberto Bassi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 SDS-PAGE analysis and identification of the phosphorylation state of protein subunits in the PSI-LHCI-LHCII-Lhcb9 supercomplex of P. patens.

a, SDS-PAGE analysis of marker (Lane 1), thylakoid (Lane 2), purified PSI-LHCI (Lane 3) and purified PSI-LHCI-LHCII-Lhcb9 (Lane 4). Each band was identified based on the mass spectrometry and proteomics analysis and indicated. b, Western-blotting analysis of thylakoid membrane, PSI-LHCI and PSI-LHCI-LHCII-Lhcb9 supercomplex using the antibodies against Lhcb9. c, Western-blotting analysis of the phosphoproteins in thylakoid membrane, PSI-LHCI and PSI-LHCI-LHCII-Lhcb9 supercomplex using a phosphothreonine antibody. d, Detection of the phosphoproteins by Pro-Q Diamond staining. The polypeptide samples were separated by SDS-PAGE and stained with Coomassie blue (left) or with Pro-Q Diamond to detect the presence of phosphoproteins (right). The amounts of the samples loaded onto each wells 2-4 were 10, 5 and 5 μg of Chls in a, b, c and d, respectively. Data presented in this figure is repeated more than three times, and all resulted in the same results.

Extended Data Fig. 2 Data collection and image processing.

a, b, Representative images of negative staining micrograph (a) and cryo-EM micrograph (b) of the PSI-LHCI-LHCII-Lhcb9 supercomplex from P. patens. The negative staining image was taken more than five times, and all resulted in similar particles. The cryo-EM micrograph was taken for 4,000 images, which were used in the subsequent structural analysis. c, Flow chart of the cryo-EM data processing for the Pp PSI-LHCI-LHCII-Lhcb9 supercomplex. d, Gold standard FSC curves of the final 3D reconstruction of the Pp PSI-LHCI-LHCII-Lhcb9 supercomplex. e, Local resolution distributions of the Pp PSI-LHCI-LHCII-Lhcb9 generated with cryoSPARC.

Extended Data Fig. 3 Cryo-EM density maps of protein subunits and representative cofactors in the PSI-LHCI-LHCII-Lhcb9 supercomplex of P. patens.

The PSI core and LHC antenna are shown as cartoon and coloured differently. Pigments (Chl a, Chl b, Bcr, Lut, Neo and Vio), lipids (1,2-dipalmitoyl-phosphatidyl-glycerol (PG), 1,2-distearoyl-monoylgalactosyl-diglyceride (MGDG), digalactosyl diacylglycerol (DGDG)) and other ligands (phylloquinone (PQN), iron/sulfur cluster (SF4) and n-dodecyl-α-D-maltoside (LMU)) are represented by sticks. The cryo-EM density maps of each subunit and cofactors are depicted in grey meshes.

Extended Data Fig. 4 Sequence alignment of Lhca1 isoforms and the assignment of the protein subunit in the structure of Pp PSI-LHCI-LHCII-Lhcb9.

a, Sequence alignment of three Lhca1 isoforms of P. patens. b, Assignment of Lhca1.2 into the structure of Pp PSI-LHCI-LHCII-Lhcb9 by comparison of the map features of characteristic residues and the corresponding sequences. Lhca1.2 contains residues Asn136, Pro170, and His233 which fit well with the density map, which excludes the existence of the other two Lhca1.1 and Lhca1.3 proteins. The red triangles show characteristic residues used for identification. Sequences are taken from Uniprot as follows: Lhca1.1[PHYPADRAFT_139567 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPADRAFT_139567 gene & protein (uniprot.org)]. Lhca1.2[PHYPADRAFT_218647 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPADRAFT_218647 gene & protein (uniprot.org)]. Lhca1.3[PHYPADRAFT_146725 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPADRAFT_146725 gene & protein (uniprot.org)].

Extended Data Fig. 5 Sequence alignment of Lhca2 isoforms and the assignment of the protein subunit in the structure of Pp PSI-LHCI-LHCII-Lhcb9.

a, Sequence alignment of five Lhca2 isoforms of P. patens. b, Assignment of Lhca2.2 by comparison of the map features of characteristic residues and the corresponding sequences. Lhca2.2 contains residues Phe69, Ala148, Pro150, Asn174, Val237, Leu252, Gly259 and Phe264 which fit well with the density map, which excludes the existence of all other Lhca2 isoform proteins. c, Assignment of Lhca2.5 by comparison of the map features of characteristic residues and the corresponding sequence. Lhca2.5 contains residues Leu69, Asn101, Tyr139, Lys140, Gln150, Arg174, Phe237, Phe252, Tyr259and Leu264 which fit well with the density map, which excludes the existence of all other Lhca2 isoform proteins. The red triangles show characteristic residues used for identification. Sequences are taken from Uniprot as follows: Lhca2.1[PHYPADRAFT_178694 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPADRAFT_178694 gene & protein (uniprot.org)]. Lhca2.2[PHYPADRAFT_151155 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPADRAFT_151155 gene & protein (uniprot.org)]. Lhca2.3[PHYPA_025494 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPA_025494 gene & protein (uniprot.org)]. Lhca2.4[PHYPADRAFT_63624 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPADRAFT_63624 gene & protein (uniprot.org)]. Lhca2.5[PHYPA_014361 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPA_014361 gene & protein (uniprot.org)].

Extended Data Fig. 6 Sequence alignment of Lhca3 isoforms and the assignment of the protein subunit in the structure of Pp PSI-LHCI-LHCII-Lhcb9.

a, Sequence alignment of Lhca3.1 and Lhca3.3 isoforms of P. patens. b, Assignment of Lhca3.1 by comparison of the map features of characteristic residues and the corresponding sequences. Lhca3.1contains unique residues Gln264, Val271, and Thr286 which fit well with the density map, which excludes the existence of the Lhca3.3 isoform proteins. The red triangles show characteristic residues used for identification. Sequences are taken from Uniprot as follows: Lhca3.1[PHYPA_009318 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPA_009318 gene & protein (uniprot.org)]. Lhca3.3[PHYPA_021044 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPA_021044 gene & protein (uniprot.org)].

Extended Data Fig. 7 Sequence alignment of Lhca2.5 and Lhca5 and the identification of Lhca2.5 subunit in the Pp PSI-LHCI-LHCII-Lhcb9 structure.

a, Sequence alignment of Lhca2.5 and Lhca5 from P. patens. Sequences are taken from the following Uniprot. b-h, Identification of the Lhca2.5 subunit in LHCI belt I of Pp PSI-LHCI-LHCII-Lhcb9 by comparison of the cryo-EM density map (b) features of characteristic amino acid residues from Lhca2.5 (green) and Lhca5 (yellow). The possibility of Lhca5 as a Lhca in the structure of the Pp PSI-LHCI-LHCII-Lhcb9 is excluded on the basis of structural features of characteristic residues (c-h), respectively. The sequences used are: Lhca2.5[PHYPA_014361 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPA_014361 gene & protein (uniprot.org)]. Lhca5[PHYPA_006083 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPA_006083 gene & protein (uniprot.org)].

Extended Data Fig. 8 Structural comparisons of Lhcb9 from P. patens with Lhcbm5 from C. reinhardtii and Lhcb6 (CP24) from P. sativum.

a, Structural comparisons of Lhcb9 from Pp PSI-LHCI-LHCII-Lhcb9 (PDB code: 7XQP, green) with Lhcbm5 from PSI-LHCI (PDB code: 7DZ7, cyan) of C. reinhardtii and Lhcb6 of PSI-LHCI (PDB code: 5XNL, blue violet) of P. sativum. The Lhcbs are shown with ribbon models, whereas the pigments and lipids are shown as sticks and coloured as that of the protein subunits respectively. Different regions between the three homologues subunits are highlighted by black dashed boxes. The phytol chains of all Chls were omitted for clarity.

Extended Data Fig. 9 Structural characterization of LHCII in Pp PSI-LHCI-LHCII-Lhcb9.

a, Cryo-EM density of LHCII trimer (Lhcbm2a, Lhcbm2b, and Lhcbm2c), viewed from the stromal side. b, Cryo-EM density map of Lhcbm2c, PsaH, PsaL, PsaO. c, Structural comparisons of Lhcbm2 from Pp PSI-LHCI-LHCII-Lhcb9 (PDB code: 7XQP, crimson) with Lhcbm1 from PSI-LHCI-LHCII (PDB code: 7DZ7, cyan) of C. reinhardtii and Lhcb2 from PSI-LHCI (PDB code: 5XNL, blue violet) of Z. mays. The Lhcbs are shown with ribbon models, whereas the pigments are shown as sticks and coloured as that of the protein subunits, respectively. Regions with difference between the three homologues subunits are highlighted by black dashed boxes. The phytol chains of all Chls were omitted for clarity. The map is shown as a semitransparent surface.

Extended Data Fig. 10 Sequence alignment of Lhcbm isoforms and the assignment of the LHCII trimer protein subunits in the structure of Pp PSI-LHCI-LHCII-Lhcb9.

a, Sequence alignment of Lhcbm isoforms of P. patens. b, Assignment of Lhcbm2 by comparison of the map features of characteristic residues and the corresponding sequences. Lhcbm2 possesses residues Thr70, Ser163, Ser121and Pro166 which fit well with the density map, which excludes the existence of all other Lhcbm proteins. The red triangles show characteristic residues used for identification. Sequences are taken from Uniprot as follows: Lhcbm1[PHYPA_022864 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPA_022864 gene & protein (uniprot.org)]. Lhcbm2[PHYPA_025699 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPA_025699 gene & protein (uniprot.org)]. Lhcbm5[PHYPA_017103 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPA_017103 gene & protein (uniprot.org)]. Lhcbm6[PHYPA_030328 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPA_030328 gene & protein (uniprot.org)]. Lhcbm7[PHYPA_007358 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPA_007358 gene & protein (uniprot.org)]. Lhcbm8[PHYPA_003988 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPA_003988 gene & protein (uniprot.org)]. Lhcbm9[PHYPA_003692 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPA_003692 gene & protein (uniprot.org)]. Lhcbm11[PHYPA_003701 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPA_003701 gene & protein (uniprot.org)]. Lhcbm13[PHYPA_006353 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPA_006353 gene & protein (uniprot.org)]. Lhcbm14[PHYPA_003990 - Chlorophyll a-b binding protein, chloroplastic - Physcomitrium patens (Spreading-leaved earth moss) - PHYPA_003990 gene & protein (uniprot.org)].

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2 and Fig. 1.

Reporting Summary

Supplementary Table 1

Mass spectroscopic data.

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Source Data Fig. 1

Unprocessed gels and western blots.

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Zhang, S., Tang, K., Yan, Q. et al. Structural insights into a unique PSI–LHCI–LHCII–Lhcb9 supercomplex from moss Physcomitrium patens. Nat. Plants 9, 832–846 (2023). https://doi.org/10.1038/s41477-023-01401-4

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