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In situ structure of the red algal phycobilisome–PSII–PSI–LHC megacomplex

Nature volume 616, pages 199–206 (2023)Cite this article

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Abstract

In oxygenic photosynthetic organisms, light energy is captured by antenna systems and transferred to photosystem II (PSII) and photosystem I (PSI) to drive photosynthesis1,2. The antenna systems of red algae consist of soluble phycobilisomes (PBSs) and transmembrane light-harvesting complexes (LHCs)3. Excitation energy transfer pathways from PBS to photosystems remain unclear owing to the lack of structural information. Here we present in situ structures of PBS–PSII–PSI–LHC megacomplexes from the red alga Porphyridium purpureum at near-atomic resolution using cryogenic electron tomography and in situ single-particle analysis4, providing interaction details between PBS, PSII and PSI. The structures reveal several unidentified and incomplete proteins and their roles in the assembly of the megacomplex, as well as a huge and sophisticated pigment network. This work provides a solid structural basis for unravelling the mechanisms of PBS–PSII–PSI–LHC megacomplex assembly, efficient energy transfer from PBS to the two photosystems, and regulation of energy distribution between PSII and PSI.

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Fig. 1: Overall structures of the PBS–PSII–PSI–LHC megacomplex from P. purpureum.
Fig. 2: PBS and PSII are linked through four linker proteins.
Fig. 3: Interaction between LCM and PSII tetramers.
Fig. 4: Structural features of the PSI–LHC supercomplex.
Fig. 5: Interaction patterns between PSII and PSI.
Fig. 6: Key pigment arrangements and possible energy transfer pathways.

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Data availability

The cryo-EM density map and atomic models generated in this study have been deposited in the Electron Microscopy Data Bank (EMDB) and the PDB for the single PBS–PSII–PSI–LHC megacomplex structure at 3.3 Å resolution (EMDB ID code 33605 and PDB 7Y5E), the double PBS–PSII–PSI–LHC megacomplex structure at 4.3 Å resolution (EMDB ID code 33669 and PDB 7Y7A), the PBS structure at 3.3 Å resolution (EMDB ID code 33605 and PDB 7Y4L), the PSII-d1-d2 structure at 3.2 Å resolution (EMDB ID code 33597), the PSII-d3 structure at 3.4 Å resolution (EMDB ID code 33568), the PSI–LHC structure at 3.6 Å resolution (EMDB ID code 33561) and the lateral hexamer structure at 6.3 Å resolution (EMDB ID code 33558 and PDB 7Y1A). Two whole artificially stitched maps have been deposited in the EMDB (EMDB ID code 33618 for single PBS–PSII–PSI–LHC and 33658 for double PBS–PSII–PSI–LHC). For the publicly available atomic models used in this study, their accession codes in the PDB have been provided in the paper.

Code availability

The code used in this study to calculate phase-residue particle sorting is available at https://github.com/chengj-dot/isSPA.

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Acknowledgements

We thank the staff at the Tsinghua University Branch of the National Protein Science Facility (Beijing) for their technical support on the cryo-EM and the high-performance computation platforms. We thank H. Deng and X. Meng in Proteinomics Facility at Technology Center for Protein Sciences, Tsinghua University, for protein MS analysis. We thank the staff at the SUSTech Core Research Facilities (Shenzhen) for their technical support in HPLC analysis. We thank J.-R. Shen from the Institute of Botany, Chinese Academy of Sciences for useful discussion. We thank Y. Yin from Plant Science Facility of the Institute of Botany, Chinese Academy of Sciences, for her excellent technical assistance on 77 K fluorescence spectra. This work was supported by the National Natural Science Foundation of China (grant nos. 32271245, 32241030 and 32071192 to S.-F.S., 32150010 to Xinzheng Z., 31825009 to H.-W.W., and 32000848 to J.M.), the National Basic Research Program (grant no. 2017YFA0504600 to S.-F.S.), the National Key R&D Program of China (grant no. 2017YFA0504700 to Xinzheng Z.), and the Xplorer Prize to H.-W.W.

Author information

Author notes

  1. These authors contributed equally: Xin You, Xing Zhang, Jing Cheng, Yanan Xiao

Authors and Affiliations

  1. State Key Laboratory of Membrane Biology, Beijing Frontier Research Center for Biological Structures, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Xin You, Jianfei Ma, Shan Sun & Sen-Fang Sui

  2. Ministry of Education Key Laboratory of Protein Sciences, Tsinghua-Peking Joint Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology, Beijing Frontier Research Center for Biological Structures, School of Life Sciences, Tsinghua University, Beijing, China

    Xing Zhang & Hong-Wei Wang

  3. National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Jing Cheng & Xinzheng Zhang

  4. School of Life Sciences, Cryo-EM Center, Southern University of Science and Technology, Shenzhen, China

    Yanan Xiao & Sen-Fang Sui

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

    Xinzheng Zhang

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  1. Xin You
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Contributions

S.-F.S. supervised the project. X.Y. and Y.X. froze samples, and performed cryo-FIB milling and the sequence analysis. Xing Z. optimized the data collection scripts. Xing Z., X.Y. and Y.X. collected the EM data. Xing Z. and J.C. performed the EM analysis under the supervision of H.-W.W. and Xinzheng Z. X.Y. performed the model building and the structure refinement. X.Y. and Y.X. performed the biochemical experiments. J.M. provided the red algae cells and did the initial tries. X.Y., Y.X., S.S. and S.-F.S analysed the structure. All authors contributed to writing the manuscript.

Corresponding authors

Correspondence to Xinzheng Zhang, Hong-Wei Wang or Sen-Fang Sui.

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Extended data figures and tables

Extended Data Fig. 1 Overview of FIB lamella sample preparation and flowchart of cryo-EM study.

a, A representative EM image of P. purpureum lamella at low magnification. 46 tilt series images were collected with similar results. b, A representative EM image of P. purpureum lamella (selected from 46) at media magnification. 46 similar tilt series were collected . c, A flowchart of the cryo-EM analysis. 23,637 sub-tomogram particles were picked out from tomograms reconstructed from 46 tilt series in emClarity. Alignment and averaging of subtomograms yielded an 8 Å structure. 3D classification showed two distinct conformations, the single and double PBS-PSII–PSI-LHC megacomplexes. The tilt images corresponding to the all subtomograms were extracted and subjected to local refinement in RELION with a soft-edge mask of PBS, yielding a 6.5 Å structure of PBS. This structure was then used as a high-resolution reference to pick out the potential particles(762,000) in high-dose image data (2,245) by method isSPA. After further particle filtering, 3D classifications and refinements, we obtained high resolution structures of single and double PBS-PSII-PSI-LHC megacomplexes from 215,000 and 87,000 particles, respectively.

Extended Data Fig. 2 Single-particle analysis of each sub-region of PBS-PSII-PSI-LHC complex.

a, Density maps of single and double PBS-PSII-PSI-LHC megacomplexes that merged from final results, demonstrating the basic structural architectures. b, Structure-refinement procedure of sub-region of PSII. c, Structure-refinement procedure of sub-region of PSI-LHC. d, Image processing of two sub-regions, a hexamer and a PSII dimer, at the center connecting position of double PBS-PSII-PSI-LHC megacomplex. e-j, Results of sub-regional refinements and the resolution estimations.

Extended Data Fig. 3 The distribution of single and double PBS-PSII-PSI-LHC megacomplexes in cell.

a, A tomogram (selected from 46) with repositioned single and double PBS-PSII-PSI-LHC megacomplexes showing the face view of PBS. Thylakoid membrane was represented as transparent density layers. b, A local area between two thylakoid membranes. Rotated 90° to present the PSII and PSI-LHCs organization in membrane. c, A tomogram (selected from 46) showing the side view of single and double PBS-PSII-PSI-LHC megacomplexes. d, Histogram of single and double PBS-PSII-PSI-LHC megacomplexes in 92 regions from 46 tomograms (each tomogram was separated into two regions).

Extended Data Fig. 4 Characterization of connector (CNT), PsbY and Psb31.

a, Structure of CNT is shown as cartoon representation. N- and C-terminal domains are colored in warmpink and green, respectively. b, Cryo-EM density (mesh) for the CNT superimposed with its atomic model (cartoon). c, Location of CNT shows that the N-terminal domain is inserted into a hydrophobic cavity created by transmembrane helices of D1, D2, CP43, PsbK, PsbJ, PsbE and PsbF. d, e, The interactions of PsbY with PsbE and PsbF. The amino acid residues involved in the interactions are shown as sticks in the enlarged boxes. f, Sequence alignment of PsbY from P. purpureum and other species. g, The interactions between PsbY, PsbF and CNT. C-/N-terminus of PsbY and PsbF are shown as surface representation. h, Cryo-EM density (mesh) for the Psb31 superimposed with its atomic model (cartoon). i, Structural comparison of Psb31 from P. purpureum and C. gracilis. The root mean square deviation between them is 1.34 Å2. j, Sequence alignment of Psb31 from P. purpureum and C. gracilis. The positively charged area of Psb31 is marked with a blue line. The extra loop of Psb31 from P. purpureum is boxed in rectangles. k, Electrostatic interaction between Psb31 and PSII core. Positively and negatively charged areas are boxed in blue and red rectangles, respectively.

Extended Data Fig. 5 Interactions between PBS and PSII.

a, Overview of LRC3, LPP1 and LPP2 interacting with both PBS and PSII from two different views. b, The interactions of LRC3 with CP43′ and PsbH. The amino acids involved in interaction are shown as sticks. c, A C-terminus shift of Psb34 occurs due to the interaction between LRC3 and Psb34. d, Electrostatic interaction of LPP1 with PBS core and PSII. e, Structural alignment of LCM and LCM′. The arrow indicates a shift of LCM-PB-loop. f, Sequence alignment of PB-loop/ LCM from different red algae and cyanobacteria. PB-loop of LCM is highlighted by a solid line. Key residues participating in interactions are marked as red triangle. Used species are red algae: Porphyridium purpureum, Griffithsia pacifica, Rhodymenia pseudopalmata and Chondrus crispus and cyanobacteria: Synechocystis sp. PCC 6803, Synechococcus sp. strain PCC 7002, Anabaena sp. PCC 7120 and Thermosynechococcus vulcanus NIES-2134. g, Overview of LCM/LCM′ and ApcD/ApcD′ interacting with PSII. h, Structural alignment of LCM and LCM′. The arrow indicates a shift of LCM-PB-loop. i, Interaction of ApcD (left), ApcD′(right) with PSII-d1 and PSII-d2. c, Structural alignment of CP43 from PSII-d1 and PSII-d2. Steric hindrance created by ApcD′ is shown as surface representation in red box. The arrow indicates the N-terminus shift of CP43/PSII-d2 due to the steric hindrance.

Extended Data Fig. 6 Structural and sequence analysis of PSI-LHC subunits.

a, Structural and sequence alignment of PsaR from P. purpureum and Chaetoceros gracilis. Left, structural alignment of PsaR from P. purpureum and C. gracilis. The Chls a and key residues are shown as sticks. The lost loop of Pp_PsaR is boxed in rectangle. Right, sequence alignment of PsaR from P. purpureum and C. gracilis. Key residues participating in Chls a binding are marked as red triangle. b, Phylogenetic analysis of LHCR and RedCAP from red algal lineage species, RedCAP from P. purpureum is boxed in red. c-d, Sequence alignments of RedCAP (c), PsaI, PsaL and PsaM (d) from P. purpureum and other algae with secondary plastids of red algal origin. Key residues participating in interactions are marked as red triangle or red line. Used species are Porphyridium purpureum, Cyanidioschyzon merolae, Phaeodactylum tricornutum, Aureococcus anophagefferens, Emiliania huxleyi, Guillardia theta and Galdieria sulphuraria.

Extended Data Fig. 7 Structural analysis of PSI-LHC.

a, The cryo-EM density map of PSI-LHC is viewed from the stromal side (left) and along the membrane plane (right) at a threshold level of 0.203. All subunits are color-coded. The density map of the boxed area is presumed to Cyt c6 according to the cryo-EM structure of Fd-PSI-c6 complex from Thermosynechococcus elongatus BP-1. b, Enlarged view of the boxed area of a shows the electrostatic interaction of Fd with PsaA, PsaC, PsaD and PsaE. c, Key sites of Fd-PSI interactions. Six negatively charged residues D29/Fd, E32/Fd, D60/Fd, D69/Fd, E95/Fd and E96/Fd are salt-bridged with positively charged residues R39/PsaE, K35/PsaC, R37/PsaA, K41/PsaA and K105/PsaD, respectively. Furthermore, an additional cation-π interaction is formed by an aromatic residue Y99/Fd with R19/PsaC. The residues involved in the interactions are shown as sticks. d, The edge-to-edge distance (Å) between [2Fe-2S] cluster of Fd and [4Fe-4S] cluster of FB in PsaC is measured (solid lines). e, f, The structures of P. purpureum PSI-LHC (e) and C. merolae PSI-LHCR (PDB entry 5ZGB) (f) are shown as cartoon representation. Compared to C. merolae PSI-LHCR, the extra parts of P. purpureum PSI-LHC are highlighted as surface representation in e. g, Structural alignment of RedCAP and Lhcr1*. N-terminal loop and AB-loop of RedCAP are boxed in rectangles. h, Detailed interactions of PsbR with Lhcr1, Lhcr6 and Lhcr7.

Extended Data Fig. 8 Possible energy transfer pathways from PBS to PSII/PSI and in PSI-LHC.

a, Overall energy transfer pathways from two pairs of energy terminal emitters (A3\({{\rm{\alpha }}}_{{\rm{ApcD}}}\), A3\({{\rm{\alpha }}}_{{\rm{ApcD}}}{\rm{{\prime} }}\), A2\({\alpha }_{{{\rm{L}}}_{{\rm{CM}}}}\) and A2\({\alpha }_{{{\rm{L}}}_{{\rm{CM}}}}{\rm{{\prime} }}\)) to reaction center of PSII (P680). b-e, Enlarged views of a show the energy transfer details from the terminal emitters to P680. The red arrows in d and e indicate that the energy further flow to PSI. f, The distribution and composition of five low energy state Chl clusters of PSI core. Chl clusters Chl1 to Chl5 are boxed in rectangles. The red arrows indicate the direction of the energy transfer. g, Possible energy transfer pathways of the PSI-LHC supercomplex viewed normal to the membrane plane from the stromal side (left) and along the membrane plane (right), respectively. Key Chl a and Bcr are shown as bold-stick and the π-π distances (Å) for the adjacent pigments are labelled in black. P700 and the low energy state Chl pairs are boxed in oval and rectangles, respectively. Possible energy transfer pathways are designated as L1RBs, L2Bs, L3As, L4Al, L5Fs, L6RBs/L6RBl, L7Bs/L7RBl and RCBl (based on Lhcr/RedCAP number, PSI core subunit, and stromal or lumenal side) and described as blow. L1RBs: Chl dimer a603/607/Lhcr1 - a201/PsaR (11.5 Å) – Chl dimer a824/825/PsaB (13.7 Å) - a826/PsaB (11.2 Å) - Chl dimer a828/838/PsaB (17.7 Å) - P700 (25.9 Å); L2Bs: Chl dimer a603/607/Lhcr2 - Chl dimer a815/823/PsaB (16.6 Å) - Chl dimer a824/825/PsaB (15.0 Å) - a826/PsaB (11.2 Å) - Chl dimer a828/838/PsaB (17.7 Å) - P700 (25.9 Å); L3As: Chl dimer a603/607/Lhcr3- Chl dimer a810/818/PsaA (16.2 Å) - Chc1 (14.7 Å) - a822/PsaA (10.6 Å) – Chc4 (16.9 Å) - P700 (25.9 Å); L4Al: Chl dimer a603/607/Lhcr4 - a805/PsaA (22.2 Å) - Chl dimer a806/807/PsaA (10.7 Å) - P700 (22.4 Å); L5Fs: Chl dimer a603/607/Lhcr5 - a302/PsaF (18.2 Å) - Chl dimer a803/301/PsaB-PsaF (14.3 Å) – A0/A (11.9 Å) - P700 (25.9 Å); L6RBs: Chl dimer a603/607/Lhcr6 - a201/PsaR (23.0 Å) - Chl dimer a824/825/PsaB (13.7 Å) - a826/PsaB (11.2 Å) - Chl dimer a828/838/PsaB (17.7 Å) - P700 (25.9 Å); L6RBl: a606/Lhcr6 - a301/PsaR (17.5 Å) - a819/PsaB (20.5 Å) - Chl dimer a836/837/PsaB (17.2 Å) - Chl dimer a828/838/PsaB (12.8 Å) - P700 (25.9 Å); L7Bs: Chl dimer a603/607/Lhcr7 - Chl dimer a815/823/PsaB (12.9 Å) - Chl dimer a824/825/PsaB (15.0 Å) - a826/PsaB (11.2 Å) - Chl dimer a828/838/PsaB (17.7 Å) - P700 (25.9 Å); L7RBl: a612/Lhcr7 - a301/PsaR (8.8 Å) - a819/PsaB (20.5 Å) - Chl dimer a836/837/PsaB (17.2 Å) - Chl dimer a828/838/PsaB (12.8 Å) - P700 (25.9 Å); RCBl: Chl dimer a606/612/RedCAP - Chl dimer a813/832/PsaB-PsaA (14.0 Å) - P700 (25.3 Å).

Extended Data Fig. 9 77 K fluorescence emission spectra of P. purpureum and pigment analysis of the thylakoid membrane from P. purpureum.

a, b, Low temperature (77 K) fluorescence emission spectra of P. purpureum cell locked in solid line. The excitation wavelength was 560 nm (a) and 440 nm (b). c, Pigment analysis by HPLC. The elutes were recorded at 445 nm. Zex, zeaxanthin; Chl a, Chlorophyll a; Bcr, β-carotene.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistic
Full size table

Supplementary information

Reporting Summary

Supplementary Table 1

Cofactors associated with each subunit of the PBS–PSII–PSI–LHC megacomplex in this study.

Supplementary Table 2

The mass spectrometric analysis of the thylakoid membrane from P. purpureum.

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You, X., Zhang, X., Cheng, J. et al. In situ structure of the red algal phycobilisome–PSII–PSI–LHC megacomplex. Nature 616, 199–206 (2023). https://doi.org/10.1038/s41586-023-05831-0

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