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Subunit and chlorophyll organization of the plantphotosystem II supercomplexLaura S. van Bezouwen1†, Stefano Caffarri2, Ravindra S. Kale3, Roman Kouřil3,Andy-Mark W. H. Thunnissen1, Gert T. Oostergetel1 and Egbert J. Boekema1*
Photosystem II (PSII) is a light-driven protein, involved in the primary reactions of photosynthesis. In plant photosyntheticmembranes PSII forms large multisubunit supercomplexes, containing a dimeric core and up to four light-harvestingcomplexes (LHCs), that act as antenna proteins. Here we solved a three-dimensional (3D) structure of the C2S2M2supercomplex from Arabidopsis thaliana using cryo-transmission electron microscopy (cryo-EM) and single particleanalysis at an overall resolution of 5.3 Å. Using a combination of homology modelling and restrained refinement againstthe cryo-EM map it was possible to model atomic structures for all antenna complexes and almost all core subunits. Welocated all 35 chlorophylls of the core region based on the cyanobacterial PSII structure, whose positioning is highlyconserved, as well as all the chlorophylls of the LHCII S- and M trimers. A total of 13 and 9 chlorophylls were identified inCP26 and CP24, respectively. Energy flow from LHC complexes to PSII reaction centre is proposed to follow preferentialpathways: CP26 and CP29 directly transfer to the core using several routes for efficient transfer; the S-trimer is directlyconnected to CP43 and the M-trimer can efficiently transfer energy to the core through CP29 and the S-trimer.
The photosynthetic reactions in plants, cyanobacteria and algaeare catalysed by four major protein complexes, photosystem I(PSI), PSII, cytochrome b6 f and ATPase. These proteins are
embedded in the thylakoid membranes, and in plants thesemembranes are located in a special organelle, the chloroplast. Theprimary photosynthetic reactions are light driven, and the harvestedenergy is used by PSI and PSII to transport electrons over themembranes and to establish a membrane proton gradient1,2.
To understand the photosynthetic reactions, detailed knowledgeabout the structure of these protein complexes is necessary. Forplant PSI high-resolution structures are known3, but for plantPSII the structural information is limited. For cyanobacteria4–6
and red algae7 there is a high-resolution PSII structure based onX-ray crystallography, but no high-resolution structure for higherplants could be obtained by protein crystallography. Very recentlythis goal was achieved by single particle cryo-EM and image processing8.
PSII is a large multisubunit protein complex containing adimeric core and a number of peripheral membrane-embeddedantenna complexes. The core complex comprises between 20 and23 protein subunits, depending on the organism. The catalyticheart of the core is the reaction centre, which is highly conservedbetween plants, algae and cyanobacteria. The reaction centreconsists of four subunits, PsbA (D1), PsbB (CP47), PsbC (CP43)and PsbD (D2), which are also the largest membrane-intrinsicsubunits. PsbA and PsbD form the photochemical reaction centrewhere the charge separation takes place as well as electron transferover the membrane. Both subunits bind in total six chlorophylls(Chls), and PsbA has in addition two pheophytins. PsbB andPsbC are the internal antenna proteins, which bind severalchlorophylls. These two subunits are involved in light harvestingand transporting excitation energy from peripheral antennasubunits towards the photochemical reaction centre1. In addition
to the reaction centre there are several other small intrinsic subunits,which are present in all organisms: PsbE, PsbF, PsbH, PsbI-M,PsbTc, PsbX, PsbY and PsbZ. These subunits are structurally andfunctionally conserved but less strongly related between organismsthan those of the reaction centre. Plant PsbY has, for instance, anadditional membrane spanning helix as compared to its bacterialcounterpart. Plants lack the cyanobacterial subunit Ycf12 but havetwo additional subunits, PsbTn and PsbW. Their location couldbe established in the plant supercomplex structure8.
PSII has a special set of three to four extrinsic subunits of theoxygen-evolving complex (OEC) which shields the water splittingmachinery9. The 33 kDa subunit PsbO, which stabilizes themanganese complex, is present in all organisms. The set ofsmaller subunits is variable. Plants have PsbP, PsbQ and PsbR10
of which PsbP and PsbQ subunits are involved in optimizing theoxygen evolution at physiological concentrations of calcium andchloride9. PsbR needs PsbJ for stabilization and seems importantfor the assembly of PsbP9. The location of the extrinsic subunitsfor plants is now largely established8.
The dimeric plant core complex (C2) can form supercomplexeswith up to six LHCII trimers, which are the major components ofthe antenna system. LHCII consists of heterotrimers composed ofdifferent combinations of Lhcb1, Lhcb2 and Lhcb311. These trimersare linked to the core complex by the minor monomeric antennaproteins Lhcb4 (CP29), Lhcb5 (CP26) and Lhcb6 (CP24)12. Atpresent, the largest purifiable PSII supercomplex, the C2S2M2 particle,binds four LHCII trimers, two strongly bound (S) and two moder-ately strongly bound (M) trimers. Two additional, loosely bound(L), trimers could be detected in spinach supercomplexes (indicatedas C2S2M2L2) and A. thaliana supercomplexes and megacomplexes13.
The light-harvesting proteins have been the subject of manystudies. Over the years several crystal structures were solved for
1Electron microscopy and Protein crystallography group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9747 AGGroningen, The Netherlands. 2Aix Marseille Université, CEA, CNRS, BIAM, Laboratoire de Génétique et Biophysique des Plantes, 13009 Marseille, France.3Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Biophysics, Faculty of Science, Palacký University, Šlechtitelů 27,783 71 Olomouc, Czech Republic. †Present address: Cryo-Electron Microscopy, Bijvoet Centre for Biomolecular Research, Faculty of Science, UtrechtUniversity, 3584 CH Utrecht, Netherlands (L.S.v.B.). *e-mail: [email protected]
ARTICLESPUBLISHED: XX XX 2017 | VOLUME: 3 | ARTICLE NUMBER: 17080
NATURE PLANTS 3, 17080 (2017) | DOI: 10.1038/nplants.2017.80 | www.nature.com/natureplants 1
Subunit and chlorophyll organization of the plantphotosystem II supercomplexLaura S. van Bezouwen1†, Stefano Caffarri2, Ravindra S. Kale3, Roman Kouřil3,Andy-Mark W. H. Thunnissen1, Gert T. Oostergetel1 and Egbert J. Boekema1*
Photosystem II (PSII) is a light-driven protein, involved in the primary reactions of photosynthesis. In plant photosyntheticmembranes PSII forms large multisubunit supercomplexes, containing a dimeric core and up to four light-harvestingcomplexes (LHCs), that act as antenna proteins. Here we solved a three-dimensional (3D) structure of the C2S2M2supercomplex from Arabidopsis thaliana using cryo-transmission electron microscopy (cryo-EM) and single particleanalysis at an overall resolution of 5.3 Å. Using a combination of homology modelling and restrained refinement againstthe cryo-EM map it was possible to model atomic structures for all antenna complexes and almost all core subunits. Welocated all 35 chlorophylls of the core region based on the cyanobacterial PSII structure, whose positioning is highlyconserved, as well as all the chlorophylls of the LHCII S- and M trimers. A total of 13 and 9 chlorophylls were identified inCP26 and CP24, respectively. Energy flow from LHC complexes to PSII reaction centre is proposed to follow preferentialpathways: CP26 and CP29 directly transfer to the core using several routes for efficient transfer; the S-trimer is directlyconnected to CP43 and the M-trimer can efficiently transfer energy to the core through CP29 and the S-trimer.
The photosynthetic reactions in plants, cyanobacteria and algaeare catalysed by four major protein complexes, photosystem I(PSI), PSII, cytochrome b6 f and ATPase. These proteins are
embedded in the thylakoid membranes, and in plants thesemembranes are located in a special organelle, the chloroplast. Theprimary photosynthetic reactions are light driven, and the harvestedenergy is used by PSI and PSII to transport electrons over themembranes and to establish a membrane proton gradient1,2.
To understand the photosynthetic reactions, detailed knowledgeabout the structure of these protein complexes is necessary. Forplant PSI high-resolution structures are known3, but for plantPSII the structural information is limited. For cyanobacteria4–6
and red algae7 there is a high-resolution PSII structure based onX-ray crystallography, but no high-resolution structure for higherplants could be obtained by protein crystallography. Very recentlythis goal was achieved by single particle cryo-EM and image processing8.
PSII is a large multisubunit protein complex containing adimeric core and a number of peripheral membrane-embeddedantenna complexes. The core complex comprises between 20 and23 protein subunits, depending on the organism. The catalyticheart of the core is the reaction centre, which is highly conservedbetween plants, algae and cyanobacteria. The reaction centreconsists of four subunits, PsbA (D1), PsbB (CP47), PsbC (CP43)and PsbD (D2), which are also the largest membrane-intrinsicsubunits. PsbA and PsbD form the photochemical reaction centrewhere the charge separation takes place as well as electron transferover the membrane. Both subunits bind in total six chlorophylls(Chls), and PsbA has in addition two pheophytins. PsbB andPsbC are the internal antenna proteins, which bind severalchlorophylls. These two subunits are involved in light harvestingand transporting excitation energy from peripheral antennasubunits towards the photochemical reaction centre1. In addition
to the reaction centre there are several other small intrinsic subunits,which are present in all organisms: PsbE, PsbF, PsbH, PsbI-M,PsbTc, PsbX, PsbY and PsbZ. These subunits are structurally andfunctionally conserved but less strongly related between organismsthan those of the reaction centre. Plant PsbY has, for instance, anadditional membrane spanning helix as compared to its bacterialcounterpart. Plants lack the cyanobacterial subunit Ycf12 but havetwo additional subunits, PsbTn and PsbW. Their location couldbe established in the plant supercomplex structure8.
PSII has a special set of three to four extrinsic subunits of theoxygen-evolving complex (OEC) which shields the water splittingmachinery9. The 33 kDa subunit PsbO, which stabilizes themanganese complex, is present in all organisms. The set ofsmaller subunits is variable. Plants have PsbP, PsbQ and PsbR10
of which PsbP and PsbQ subunits are involved in optimizing theoxygen evolution at physiological concentrations of calcium andchloride9. PsbR needs PsbJ for stabilization and seems importantfor the assembly of PsbP9. The location of the extrinsic subunitsfor plants is now largely established8.
The dimeric plant core complex (C2) can form supercomplexeswith up to six LHCII trimers, which are the major components ofthe antenna system. LHCII consists of heterotrimers composed ofdifferent combinations of Lhcb1, Lhcb2 and Lhcb311. These trimersare linked to the core complex by the minor monomeric antennaproteins Lhcb4 (CP29), Lhcb5 (CP26) and Lhcb6 (CP24)12. Atpresent, the largest purifiable PSII supercomplex, the C2S2M2 particle,binds four LHCII trimers, two strongly bound (S) and two moder-ately strongly bound (M) trimers. Two additional, loosely bound(L), trimers could be detected in spinach supercomplexes (indicatedas C2S2M2L2) and A. thaliana supercomplexes and megacomplexes13.
The light-harvesting proteins have been the subject of manystudies. Over the years several crystal structures were solved for
1Electron microscopy and Protein crystallography group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9747 AGGroningen, The Netherlands. 2Aix Marseille Université, CEA, CNRS, BIAM, Laboratoire de Génétique et Biophysique des Plantes, 13009 Marseille, France.3Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Biophysics, Faculty of Science, Palacký University, Šlechtitelů 27,783 71 Olomouc, Czech Republic. †Present address: Cryo-Electron Microscopy, Bijvoet Centre for Biomolecular Research, Faculty of Science, UtrechtUniversity, 3584 CH Utrecht, Netherlands (L.S.v.B.). *e-mail: [email protected]
ARTICLESPUBLISHED: XX XX 2017 | VOLUME: 3 | ARTICLE NUMBER: 17080
NATURE PLANTS 3, 17080 (2017) | DOI: 10.1038/nplants.2017.80 | www.nature.com/natureplants 1
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
SUPPLEMENTARY INFORMATIONVOLUME: 3 | ARTICLE NUMBER: 17080
NATURE PLANTS | DOI: 10.1038/nplants.2017.80 | www.nature.com/natureplants 1
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Supplementary Information. Van Bezouwen et al. Nature Plants (2017)
Supplementary Figure 1 │ Original EM images. Images are sums of frames corrected for beam induced motion and drift. (a) Image taken at a defocus of 1.2 μm. (b) Image taken at a defocus of 3.0 μm. Some side views (white arrowheads) and top views (black arrowheads) are marked.
Supplementary Figure 2 │ Angular distribution of angles of cryo-EM projections with respect to the 3D model. The length and the colour of the bars represent the population of different projection directions. The 3D model is seen from aside.
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
NATURE PLANTS | DOI: 10.1038/nplants.2017.80 | www.nature.com/natureplants 2
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Supplementary Figure 3 │ A gallery of 2D averages that reconstitute the final cryo-TEM map. 16 2D maps show the PSII supercomplex in top-view, side-views and intermediate views.
Supplementary Figure 4 │ Local resolution of the cryo-EM density map. (a) Slice through the PSII supercomplex in the centre of the membrane plane to show the differences in local resolution. At the core region the resolution is at its best. The S-LHCII trimer and CP26 have the highest resolution for the antenna proteins. The M-LHCII trimer and the CP24 have the lowest resolution of the supercomplex. Details in the detergent shell were not resolved. (b) Resolution inside the protein. The extrinsic part is well resolved. The helices inside the membrane of the core have the highest resolution.
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
NATURE PLANTS | DOI: 10.1038/nplants.2017.80 | www.nature.com/natureplants 3
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Supplementary figure 5 | FSC curve of the map vs model. The resolution is 7.7 Å, based on the 0.5 criterion. This is lower than the final resolution 5.3 Å in figure 1c, because there are large resolution differences within the map. In addition, there are areas left empty, where most likely pigments and lipids are present, due to limited resolution.
Supplementary Figure 6 │ The region near the expected location of the manganese cluster in plants. Subunits PsbA (purple), PsbB (cyan), PsbD (blue) and PsbO (yellow) are shown and their expected coordination of the manganese cluster (manganese yellow, calcium green and waters in red). There is no density present for the manganese cluster. The C-terminus of PsbA is highly disordered, a slight disorder at the C-terminus of PsbD, and there is a disorder at the loop of PsbO. The regions of PsbA and PsbD interact with manganese cluster and the loop of PsbO interact with the C-terminus of PsbA in cyanobacteria. The density for these regions is missing in the cryo-EM map.
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NATURE PLANTS | DOI: 10.1038/nplants.2017.80 | www.nature.com/natureplants 4
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Supplementary Figure 7│Fit of the antenna subunits inside the cryo-EM structure. (a) Fit of a Lhcb1 subunit of the S-trimer, (b) Fit of a Lhcb1 subunit of the M-trimer, (c) Fit of CP26, (d) fit of CP29 and (e) fit of CP24.
Supplementary Figure 8│Comparison of the 3D cryo structure with a negative stain 2D projection map. (a) The 3D cryo-EM density map, showing all the main regions. Color coding is the same as in Fig. 2. (b) 2D projection map of the C2S2M2 particle20. The core is from cyanobacteria64 and the Lhcb subunits are from spinach12 for both trimeric LHCII (blue and green) and monomeric Lhcb (red, yellow and pink). The LHCII-S and LHCII-M trimer are contoured in light green. Note that the exact orientation of CP26 and CP29 is slightly different in the two models, while for the orientation of CP24 is significantly different. The scale bar is 100 Å.
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
NATURE PLANTS | DOI: 10.1038/nplants.2017.80 | www.nature.com/natureplants 5
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