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Structure of spinach photosystem II–LHCII supercomplex at 3.2 Å resolution

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Abstract

During photosynthesis, the plant photosystem II core complex receives excitation energy from the peripheral light-harvesting complex II (LHCII). The pathways along which excitation energy is transferred between them, and their assembly mechanisms, remain to be deciphered through high-resolution structural studies. Here we report the structure of a 1.1-megadalton spinach photosystem II–LHCII supercomplex solved at 3.2 Å resolution through single-particle cryo-electron microscopy. The structure reveals a homodimeric supramolecular system in which each monomer contains 25 protein subunits, 105 chlorophylls, 28 carotenoids and other cofactors. Three extrinsic subunits (PsbO, PsbP and PsbQ), which are essential for optimal oxygen-evolving activity of photosystem II, form a triangular crown that shields the Mn4CaO5-binding domains of CP43 and D1. One major trimeric and two minor monomeric LHCIIs associate with each core-complex monomer, and the antenna–core interactions are reinforced by three small intrinsic subunits (PsbW, PsbH and PsbZ). By analysing the closely connected interfacial chlorophylls, we have obtained detailed insights into the energy-transfer pathways between the antenna and core complexes.

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Figure 1: Single-particle cryo-EM analyses of spinach PSII–LHCII supercomplex.
Figure 2: Overall architecture of spinach PSII–LHCII supercomplex.
Figure 3: The extrinsic subunits of spinach PSII.
Figure 4: Antenna–core and antenna–antenna interactions in the PSII–LHCII supercomplex.
Figure 5: Energy transfer pathways from antenna complexes to reaction centres.

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

The cryo-EM map of the spinach PSII–LHCII supercomplex has been deposited in the Electron Microscopy Data Bank with accession code EMD-6617. The corresponding structure model has been deposited in the Protein Data Bank under accession code 3JCU.

Change history

  • 01 June 2016

    The received date year was corrected from 2015 to 2016.

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Acknowledgements

We thank J. P. Zhang and X. L. Zhao for their assistance in preparing thylakoid samples. Cryo-EM data collection was carried out at the Center for Biological Imaging, Core Facilities for Protein Science at the Institute of Biophysics (IBP), Chinese Academy of Sciences (CAS), and at the National Center for Protein Science Shanghai (NCPSS), Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences/Shanghai Science Research Center, Chinese Academy of Sciences, Shanghai, China. We thank X. J. Huang, G. Ji, W. Ding, F. Sun, and other staff members at the Center for Biological Imaging (IBP, CAS); L. L. Kong, X. Y. Shi, Y. N. He, J. P. Ding, and M. Lei for their support during data collection; X. B. Liang and X. M. An for support in organizing data collection trips; F. L. Zhang, J. Zhou, and Y. Li for support in measuring the oxygen evolution activity; L. L. Niu and X. Ding for mass spectrometry; J. H. Li for assistance in fluorescence measurement; R. Bassi, A. Pinnola and R. Croce for sharing experiences in purifying plant PSII–LHCII supercomplexes; and Y. Xiang for advice on cryo-EM sample preparation and structure refinement. The project was funded by National 973 project grant 2011CBA00900, the Strategic Priority Research Program of CAS (XDB08020302) and National Natural Science Foundation of China (31570724, 31270793 and 31170703). Z.L. and X.Z. received scholarships from the ‘National Thousand (Young) Talents Program’ from the Office of Global Experts Recruitment in China.

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Contributions

X.W., X.S., P.C. and X.L. purified the spinach PSII–LHCII supercomplex; M.L. and P.C. characterized the spectroscopic features, protein and pigment contents, and oxygen-evolving activity of the samples; X.W., X.S. and X.Z. collected and processed cryo-EM data; X.Z. reconstructed the 3.2 Å resolution map and supervised cryo-EM structure determination; X.W. and Z.L. built and refined the structure model; X.W., M.L., X.Z. and Z.L. analysed the structure; Z.L. and W.C. conceived and coordinated the project; and the manuscript was written by X.W., M.L., X.Z. and Z.L.

Corresponding authors

Correspondence to Mei Li or Xinzheng Zhang or Zhenfeng Liu.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks Roberta Croce, Jian-Ren Shen and Thomas Walz for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Purification and characterization of the spinach PSII–LHCII supercomplex.

a, Sucrose gradient of solubilized grana membranes. The membrane preparations were first washed with 1 mM (left) or 5 mM (right) EDTA before being solubilized by α-DDM for further purification through sucrose-gradient ultracentrifugation. The content of each band is indicated based on the absorption spectrum and SDS–PAGE results, and by comparing to previously published data. The B9 fraction obtained from the grana membrane washed with 1 mM EDTA was used for cryo-EM. Note that the grana membranes washed with 1 mM EDTA yielded less B5, B6 and B7 than the sample treated with 5 mM EDTA. b, SDS–PAGE analysis of the sucrose gradient fractions. The protein composition of each Coomassie band was indicated based on the mass spectrometry and proteomics data analysis. For gel source data, see Supplementary Fig. 1. c, Room-temperature absorption spectrum of B9 sample used for cryo-EM. Its spectrum (B9 1 mM) is compared to those of B7 (dimeric PSII core without LHCII attached; B7 5 mM) and B9 samples (B9 5 mM) fractionated from grana washed with 5 mM EDTA. Note that B9 from grana membranes washed with 1 mM EDTA showed higher peaks at 470 and 650 nm, indicating that this fraction contains higher Chl b content (from LHCIIs) than the other two. The spectra are normalized to the maximum in the red region. d, Fluorescence emission spectra of B9 sample measured at room temperature. The maximum emissions were at 681 nm (upon excitation of Chl a at 436 nm), 680 nm (upon excitation of Chl b at 473 nm) and 681 nm (upon excitation of carotenoids at 500 nm). Overlapping of these three spectra suggests that nearly all pigments in the B9 sample are well coupled and no free pigments are present. e, Pigment content analysis of B9 sample by HPLC. Based on the characteristic absorption spectrum of each peak fraction, the six major pigment peaks separated from the B9 sample are identified as neoxanthin (Neo), violaxanthin (Vio), lutein (Lut), Chl b, Chl a and β-carotene (β-car).

Extended Data Figure 2 Evaluation of the resolution of the cryo-EM structure of the spinach PSII–LHCII supercomplex.

a, Fourier shell correlation (FSC) plots. Blue, gold-standard FSC curve with a value of 0.143 at 3.2 Å resolution; red, FSC curve calculated between the cryo-EM map and the refined structure model of the PSII–LHCII supercomplex. The map-model FSC has a value of 0.5 at 3.3 Å resolution. b, Local resolutions of the cryo-EM map of the spinach PSII–LHCII supercomplex estimated by Resmap. Top, side view along the membrane plane with the luminal domain facing upwards. Bottom, bottom view from the luminal side and approximately along the membrane normal (or C2 axis). c, The statistics of the structural model of the spinach PSII–LHCII supercomplex refined against the 3.2 Å resolution cryo-EM map.

Extended Data Figure 3 Structures of the large and small intrinsic subunits of spinach PSII.

a, The four large intrinsic subunits of the spinach PSII core superposed on the corresponding subunits of the TvPSII core. The protein backbones and cofactors are shown as ribbon and stick models, respectively. Silver, spinach PSII core subunits; green, TvPSII core subunits. b, The locations of 12 low-molecular-mass intrinsic subunits in the spinach PSII–LHCII supercomplex. These subunits are coloured and the rest of the supercomplex is grey. c, The densities for the low-molecular-mass intrinsic subunits are shown as blue meshes. The corresponding models are shown as cyan sticks.

Extended Data Figure 4 Cryo-EM densities and structures of the extrinsic subunits.

a, Cryo-EM densities of PsbO, PsbP, PsbQ and PsbTn. b, The binding sites of spinach PsbP, PsbQ and PsbO compared to those of the extrinsic subunits in CcPSII and TvPSII. The spinach PSII core is shown at an angle identical to that of the CcPSII/TvPSII core. PDB codes: 4YUU (CcPSII); 3WU2 (TvPSII). c, Superposition of PsbP bound in the supercomplex with the isolated PsbP. Colour code: yellow, PsbP in the supercomplex; blue, isolated PsbP (PDB code: 4RTI). Loop 3A and Loop 4A indicate the loop regions between Lys90 and Ala111 and between Arg134 and Gly142, respectively. Note the conformational change in Loop 3A (arrow) when PsbP binds to the PSII core. d, Structure of PsbQ bound in the supercomplex superposed with the isolated PsbQ. Green, PsbQ in the supercomplex; red, isolated PsbQ (PDB code: 1VYK). Note the conformational change in the elongated N-terminal region from a folded state to an extended form (arrow) when PsbQ binds to the PSII core.

Extended Data Figure 5 Cryo-EM density and structure of LHCII.

a, Cryo-EM densities of the LHCII trimer in the supercomplex. Stereo pairs are shown and the view is along the membrane plane. b, Superposition of the cryo-EM structure of an LHCII monomer with the previous crystal structure (PDB code: 1RWT). The protein backbone is shown as ribbon diagrams and the cofactors are displayed as stick models. Green, cryo-EM structure; yellow, crystal structure.

Extended Data Figure 6 Cryo-EM density and structure of CP29.

a, Stereo image of the cryo-EM density of CP29 bound in the PSII–LHCII supercomplex. b, Superposition of cryo-EM structure of full-length CP29 with the previous crystal structure. Note: Chls a601 and a616 are newly observed in the cryo-EM structure of CP29. Chl a601 might account for the electron density of Chl a615 observed in the crystal structure of spinach CP29 (ref. 14). Compared to Chl a601, a615 is much closer to a611 owing to the loss of the N-terminal domain caused by proteolysis. Chl b614 is a peripheral chlorophyll found in the crystal structure, but is probably lost during purification and therefore not observed in the cryo-EM structure. Orange, cryo-EM structure; cyan, crystal structure. c, Superposition of CP29 (orange) with the structure of an LHCII monomer (green). For the cofactors, only Chl a601 is shown; the others are omitted for clarity. d, Cryo-EM density of Chl a601 in CP29. e, Cryo-EM density of Chl a616 at the interface between CP29 and CP47. f, Superposition of Lhca3 and Lhca4 structures with that of CP29 in the PSII–LHCII supercomplex. Chl a616 (CP29) and a617 (Lhca3/4) molecules are shown as stick models; the other cofactors are omitted for clarity. Orange, CP29; magenta, Lhca3; blue, Lhca4. PDB codes: 4XK8, Lhca3 and Lhca4 from the PSI–LHCI supercomplex; 1RWT, LHCII.

Extended Data Figure 7 Cryo-EM density and structure of CP26 bound in the PSII–LHCII supercomplex.

a, Stereo images of the density and overall structure of CP26. The density is shown as grey meshes and the model is in purple. The protein backbone is shown as a ribbon model; the cofactors are presented as stick models. b, The densities for Chl b601, Chl a604, Chl b607 and Chl b608 in CP26. These four chlorophylls were not predicted in the previous work, but are clearly present in the structure. c, The density for three carotenoids in CP26. Note that the density for the epoxidized head group of neoxanthin is clearly visible, while the rest of it is fairly weak (presumably owing to low occupancy or high flexibility).

Extended Data Figure 8 Cryo-EM densities of various cofactors bound in the spinach PSII–LHCII supercomplex.

a, The densities of chlorophylls, carotenoids, Mn4CaO5 and plastoquinone molecules. b, The potential lipid densities at the interfacial regions between adjacent antenna complexes. The interfaces between LHCII and PsbW, CP26 and CP43, and LHCII and CP29 are shown from left to right. Red arrows indicate the positions of potential lipid densities. The cryo-EM densities are displayed as grey meshes and the atomic models for interpretation of the densities are shown as sticks and bullets.

Extended Data Table 1 Cofactors located within each monomer of the spinach PSII–LHCII supercomplex
Extended Data Table 2 Pigment binding sites of spinach LHCII, CP29 and CP26 in the PSII–LHCII supercomplex

Supplementary information

Supplementary Figure 1

This file contains the source data for Extended Data Figure 1b. The boxed area is cropped and displayed in the Extended Data Figure 1b. (PDF 140 kb)

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Wei, X., Su, X., Cao, P. et al. Structure of spinach photosystem II–LHCII supercomplex at 3.2 Å resolution. Nature 534, 69–74 (2016). https://doi.org/10.1038/nature18020

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