Structure of the intact C2S2-type Photosystem II-LHCII supercomplex from Arabidopsis thaliana at 2.44 Å

Structure of the intact C2S2-type Photosystem II-LHCII supercomplex from Arabidopsis thaliana at 2.44 Å | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Structure of the intact C2S2-type Photosystem II-LHCII supercomplex from Arabidopsis thaliana at 2.44 Å Johannes Messinger, Jack Forsman, André Graca, Abuzer Aydin, Michael Hall, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5656066/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Photosystem II (PSII) is a large membrane-bound protein complex that is essential for oxygenic photosynthesis in plants and cyanobacteria. While there are several high-resolution structures of PS II from cyanobacteria, very few PS II structures have been reported from higher plants. Here, we present the first high-resolution structure of an intact and active PS II from Arabidopsis thaliana at a resolution of 2.44 Å, which to date represents the highest resolution structure of PS II from any land plant. The improved resolution allowed for the modeling of cofactors which have not been previously seen in higher plant PS II structures. Importantly, we were able to identify many water molecules within the PS II structure, including waters around the water-splitting manganese cluster, in the bottleneck regions of the water/proton channels, and near the non-heme iron on the acceptor side. Structural differences between cyanobacterial and plant PS II are discussed. Biological sciences/Plant sciences/Photosynthesis/Photosystem II Biological sciences/Biological techniques/Structure determination/Electron microscopy/Cryoelectron microscopy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Photosystem II (PS II) is a large protein complex located in the thylakoid membranes of oxygen-evolving photosynthetic organisms. PS II absorbs light energy and uses it to perform charge separation reactions that drive the oxidation of water and the reduction of plastoquinone 1 , 2 . This reaction results in the production of molecular oxygen, making it essential to life on Earth. The D1 and D2 subunits in the PS II core coordinate the cofactors required to perform water splitting. This includes the chlorophyll (P680) and pheophytin (Pheo) molecules, between which the primary charge separation occurs; and the manganese, calcium, oxygen cluster (Mn 4 CaO 5 cluster) that oxidizes water (Fig. 1 A) 2 . The energy required to perform water splitting is primarily captured, from sunlight, by peripheral antenna proteins and funneled toward the core antenna subunits CP43 and CP47, before being passed to P680 3 . In addition, there are several small transmembrane proteins that support the function of PS II in various ways, and the extrinsic subunits which stabilize the Mn 4 CaO 5 cluster 4 , 5 , 6 . The Mn 4 CaO 5 cluster is sequentially oxidized by successive charge separations between P680 and Pheo, facilitating the binding, de-protonation and oxidation of the two substrate water molecules. This process, known as the Kok-cycle 7 , transitions the cluster from its most reduced state, S 0, through the intermediate states S 1 , S 2 , S 3, and the transient S 4 state, leading to the formation of molecular oxygen during the S 4 ◊S 0 transition 1 , 2 , 8 . The Mn 4 CaO 5 cluster is connected to the water phase of the lumen via channels 9 . These channels allow water to access the Mn 4 CaO 5 cluster and protons to egress. Recently, it has been shown that a specific arrangement of the water network around the Mn 4 CaO 5 cluster is required for efficient water insertion and proton release during the S 2 ◊S 3 and the S 3 ◊S 0 transitions 10 – 15 . Consequently, the water network around the Mn 4 CaO 5 cluster and the proton gate must be carefully regulated to prioritize the specific conformations required for these events to occur. Across all photosynthetic organisms, the structures of the core subunits are highly conserved (D1, D2, CP43 and CP47) 16 , 17 . However, a notable difference exists near the Mn 4 CaO 5 cluster at position 87 of the D1 protein, where an alanine residue is found in plants and an asparagine residue is present in the majority of cyanobacteria, affecting the water and H-bonding networks in ways that remain to be explored 18 . Additionally, different photosynthetic species exhibit significant variation in the small transmembrane and extrinsic proteins 4 , 5 , 19 , which may alter the channels to the Mn 4 CaO 5 cluster and thereby, these networks 9 . The ability of PS II from thermophilic cyanobacteria to readily form crystals allowed several high-resolution x-ray diffraction structures of PS II to be collected from these organisms ( Thermosynechococcus vestitus or Thermosynechococcus vulcanus ) with resolutions up to 1.89 Å 20 – 23 . Recent improvements to cryo-electron microscopy (cryo-EM) have also resulted in several high-resolution structures of PS II from cyanobacteria with resolutions up to 1.71 Å 24 – 26 . However, the structure of PS II from land plants has been more difficult to obtain. Low/medium-resolution structures of PS II are available from Spinacia oleracea (spinach) (3JCU) 27 , Pisum sativum (pea) (5XNL) 28 and Picea abies (spruce) (8C29) 29 , with resolutions of 3.2 Å, 2.7 Å and 2.8 Å, respectively. Additionally, our research group has previously published a structure of PS II from Arabidopsis thaliana (7OUI) 30 at a resolution of 2.79 Å. However, this structure lacked the PsbP, PsbQ and PsbJ subunits, and the Mn 4 CaO 5 cluster. Here, we present a fully intact C 2 S 2 -PS II:LHC II supercomplex from Arabidopsis thaliana at a resolution of 2.44 Å. Results C 2 S 2 -type Photosystem II map and model The Coulomb potential map of the C 2 S 2 -type PS II was generated from 72,301 particles with enforced C2 symmetry and reached a global resolution of 2.44 Å, ranging from 2 Å resolution in the core to 3.5 Å resolution in the flexible peripheral regions (Fig. 1 B-D). The map showed clear densities for all the PS II core subunits and all extrinsic subunits, as well as for all metal ions and water ligands of the Mn 4 CaO 5 cluster (Supplementary Fig. 1A). The Mn-Mn and Mn-Ca distances in the Mn 4 CaO 5 cluster are elongated in comparison with XFEL structures 22 , 23 , suggesting partial reduction of the Mn ions during data collection (micrographs collected at 40 e − /Å −2 ). In contrast, the proteins appear practically unchanged; the disulfide bond in PsbO protein, which breaks at high electron dose (83 e − /Å −2 ) 24 , is intact in our structure (Supplementary Fig. 1B). The strongly bound LHC II trimers were successfully modeled (Fig. 1 B); however, the map showed no density for the mediumly-bound LHC II trimers in either the 3D structure or the 2D classes. For subunits with multiple similar isoforms, the model displays the best fitting isoform (Supplementary Fig. 2), although the map may contain contributions from multiple different isoforms. Cofactors Our map showed a clear density at the monomer:monomer interface which we modelled as a β-carotene (Fig. 2 A). This β-carotene molecule (named PsbI:BCR34) was found close to the PsbI subunit in both monomers. Other PS II cryo-EM maps either show no feature at this position (spinach, 3JCU), or the electrostatic potential is filled with a steric acid ( T. vestitus , 9EVX), a lineolenic acid (spruce, 8C29), or a distearoyl-monogalactosyl-diglyceride (LMG) (pea, 5XNL). Our new β-carotene model fits reasonably well into the electron/charge densities from the other maps, suggesting that this cofactor may also be present in the other species (Supplementary Fig. 3). At the closest point PsbI:BCR 34 is 5.2 Å from chlorophyll a 410 in the D1 subunit, (Fig. 2 A). It is therefore feasible that this β-carotene may interact with this chlorophyll as either an antenna or a quencher. All other β-carotene molecules in our structure can also be found in the T. vestitus (9EVX), pea (5XNL), spinach (3JCU) or spruce (8C29) structures (Fig. 2 B). Our new map also allowed the lipids digalactosyl diacylglycerol (DGD) and sulfoquinovosyl diacylglycerol (SQD) to be modelled at the interface between PS II and the strongly-bound LHC II trimer (Fig. 2 C). These lipids presumably help to anchor the strongly-bound LHC II trimers to the PSII complex in plants. In the spinach structure (3JCU) there were no cofactors modelled at these positions; however, in the pea (5XNL) and spruce (8C29) structures, there were alternate lipids modelled at these positions. Fitting SQD and DGD into the pea or spruce maps or fitting of the alternate cofactors into the Arabidopsis map resulted in poor fits (Supplementary Fig. 4–5). An α-tocopherol molecule is modelled in this region in the spruce structure (8C29). We also observe a density at a similar position which fits α-tocopherol; however, without experimental results to confirm the presence of an α-tocopherol molecule in Arabidopsis PS II, we chose not to include it in our model (Supplementary Fig. 6). Our structure includes 1202 modelled water molecules, in comparison to 1076 waters in pea (5XNL), 280 in spruce (8C29) and none in spinach (3JCU). Excluding water molecules in the LHCII trimers, our structure resolves 1040 water molecules; approximately half of those identified in the current highest resolution structure of PS II from the cyanobacterium T. vestitus 26 . . Water networks at the PS II acceptor side At the acceptor side of PSII, a water network has been identified in cyanobacterial structures that connects the stroma with the Q B site 20 , 21 , 26 . Our Arabidopsis structures finds several waters at similar positions (Fig. 3 A), as well as water positioned between Q B , D1:Tyr246 and D1:His215 (W63 in Fig. 3 A). A water in this position has been hypothesized to participate in the hydrogen-bonding network which re-protonates the D1:His215 residue following donation of a proton to Q B 31 – 34 . A water in a similar position was also found in the recent high-resolution structure from T. vestitus 26 , suggesting that this re-protonation mechanism is conserved between cyanobacteria and plants. Alignment of our PS II structure to the structure from T. vestitus (9EVX) showed that the D1 loop region, which forms part of the Q B binding pocket (D1:247–267), is shifted in the Arabidopsis structure (Fig. 3 B). Additionally, the side chain of the D1:Ile248 residue is flipped relative to the T. vestitus structure, creating space to accommodate two additional water molecules (Fig. 3 B). This loop shift is consistent with molecular dynamics (MD) simulations 35 , which indicate that this movement, (triggered by the release of Q B H 2 from the Q B pocket), can expand a water channel leading from the Q B pocket to the stroma (Supplementary Fig. 7A-B). B-factor analysis of the acceptor side region of PS II indicates that in our structure the B-factor of Q B is approximately 5 times higher than Q A (Fig. 3 C), while in the T. vestitus structure the B-factor of Q B is only twice as high as Q A 26 . Further analysis confirms the reduced occupancy of Q B in our structure (Supplementary Fig. 7C). The observed structural differences between Arabidopsis and T. vestitus are likely a result of the different light conditions during the sample preparation. Since the Arabidopsis PS II grids were prepared in dim light, a fraction of the Arabidopsis PS II particles would have an empty Q B binding pocket due to Q B reduction. Thus, the Arabidopsis Coulomb potential map around the Q B pocket likely represents an average position between the occupied Q B pocket and the empty Q B pocket conformations 35 . Water networks around the MnCaO cluster Excluding the water ligands to the Mn 4 CaO 5 cluster (W1-W4), fourteen water molecules were modelled in our structure within 9 Å of the Mn 4 CaO 5 cluster (Fig. 4 A). Nearly all these waters were clustered near the entrances of the O1, O4 and Cl1 channels (Fig. 4 ), which may suggest that having ordered water molecules in these positions is crucial for function. All the water molecules had an equivalent water in the T. vestitus structure, except for the water modeled near the D1:Ala87 residue (W200), which is in the position of the polar headgroup of the D1:Asn87 side chain of cyanobacteria. This suggests that in plants, a water binds in place of the bulky asparagine side chain, fulfilling the role of the asparagine headgroup within the hydrogen-bonding network (Supplementary Fig. 8). Random noise begins to appear outside of the structure when the contour level of the locally sharpened map is reduced below 1.29 RMSD. At this contour level the map only shows density for two of the waters in the water wheel (Fig. 4 B). However, a contour level of 0.49 RMSD there is a clear ring of density at the site of the water wheel (Fig. 4 C). As the local resolution in the core is considerably higher than the global resolution, we felt confident in modelling these waters (Fig. 1 C-D). This finding shows that the water wheel is conserved among species and supports the proposal that it plays a crucial role in water oxidation 11 , 14 , 36 . Water/proton channels to the MnCaO cluster The channels leading to the Mn 4 CaO 5 cluster, and their water and hydrogen-bonding networks are pivotal for the stability and function of the oxygen evolving complex, as they regulate water access, enable controlled proton egress, and facilitate substrate water binding 9 . Comparing them in detail across species is crucial for understanding their specific functions and design principles. Here, we extend a previous study 9 by comparing the channels in our new Arabidopsis data with those in T. vestitus and analyzing the positions of identified water molecules. O1 channel It has been hypothesized that substrate water molecules reach the Mn 4 CaO 5 cluster via the O1 channel 9 , 14 , 22 . CAVER analysis showed that the O1 channel has three branches in the Arabidopsis structure, compared to two in T. vestitus , and that these branches follow different routes to the surface of the protein (Fig. 5 A). All branches of the O1 channel in Arabidopsis pass through a common bottleneck region near the Mn 4 CaO 5 cluster. The only water molecule to be modelled in the O1 channels in the Arabidopsis structure was located in this bottleneck region (Fig. 5 B). This indicates that this region is highly ordered, while the other water molecules in the O1 channel appear to be comparatively mobile, and thus more challenging to detect at our current resolution. The importance of this bottleneck is further highlighted by its conservation across species, as a corresponding water molecule was also found in the cryo-EM structures from T. vestitus (9EVX) and in pea (5XNL) (Fig. 5 B), as well as the x-ray crystallography structures from T.vestitus 13 (7rf1) and Thermosynechococcus vulcanus 8 (6dhe). It has been proposed that this bottleneck region can act as a ‘water valve’ that can open and close via allosteric actions at desired times during water oxidation, regulating water access to the Mn 4 CaO 5 cluster 36 , 37 . The conservation of the bottleneck region and the relatively immobile water within the bottleneck support this hypothesis. However, the model of PS II from Synechocystis sp. PCC 6803 shows a different side chain orientation for D1:Glu329 and no water is modelled at the position shown in Fig. 5 B 25 . Cl1 channel The Cl1 channel has been demonstrated to facilitate proton removal during water oxidation 10 , 13 , facilitating proton transfer via the residues D1:Asp61, D1:Glu65 and D2:Glu312 9,13,23 . CAVER analysis showed that the Cl1 channel in Arabidopsis is highly similar to the Cl1 channel in T. vestitus , conserving the positions of the critical proton-transfer residues and water molecules (Fig. 6 A). This strong conservation indicates that the regulation of proton egress via the proton gate of the Cl1 channel is highly critical for water oxidation. Theoretical analyses and one experimental study indicate that the bottleneck residues and the D1-D61 side chain restrict water access to the Mn 4 CaO 5 cluster through the Cl1 channel 9 , 10 , 12 , 13 , 39 – 41 . O4 channel The O4 channel has been hypothesized to be involved in the removal of protons from the Mn 4 CaO 5 cluster during the S 0 ◊S 1 transition 9 , 14 , 37 , 38 However, it has also been suggested to supply water to the Mn 4 CaO 5 cluster 42 . The O4 channels in Arabidopsis and T. vestisus initially follow a similar path; however, they diverge when the O4 channel passes through the extrinsic proteins, which differ between plants and cyanobacteria. Five water molecules were modeled within the O4 channel of Arabidopsis. These were not limited to one bottleneck region but were instead found at various places along the channel. This is consistent with previous observations made with cyanobacterial structures that the entire O4 channel is relatively narrow 9 . 43 . In Arabidopsis, the bottleneck of the channel has a diameter of 1.6 Å compared to an average diameter of only 3 Å, indicating water mobility is restricted along the entire channel length in the O4 channel, as compared to a specific ‘water valve’ in the O1 channel. This narrow width is expected to restrict water motion and, thus, the O4 channel appears better suited for proton transfer than water delivery. Discussion The structure presented here represents a significant improvement over previously published structures of higher plant PSII, both in resolution and completeness, including our previous Arabidopsis PS II structure (7OUI) 30 . By omitting digitonin and lowering the pH during PS II extraction, we were able to obtain intact PS II particles, leading to a complete structure of active, mature PS II. It is interesting to note that the PS II particles used for the spruce structure (8C29) were isolated at pH 7.5 and lacked PsbJ, PsbP, PsbQ and the Mn 4 CaO 5 cluster, despite the absence of digitonin, suggesting that the choice of pH is critical for retaining the extrinsic subunits and the Mn 4 CaO 5 cluster. In all the higher plant structures, including the one presented here, the lipids connecting the CP47 subunit to the s-LHC II trimer had a different identity. As the identity of the lipids at this position may affect the strength of the LHC II trimer binding to the core and since LHC II trimers can dissociate from the core in high light conditions 44 , we hypothesize that the identity of these lipids may modulate this protective mechanism. Simulations of the acceptor side of PS II provide evidence that the structure of the Q B pocket changes when it is vacated 35 . Compared to the T. vestitus structure (9EVX) our Arabidopsis structure had a shifted position for the D1:247–267 region, along with unusually high B-factors. This is likely the result of combining data of PSII particles that have Q B bound with a significant fraction of particles lacking Q B . This finding supports calculations showing movement of this D1 loop region following the dissociation of Q B H 2 from the Q B binding pocket 35 . Our present data is most consistent with one substrate water delivery channel, the O1 channel, and two proton release channels, one facilitating proton release during the S 0 -S 1 transition (O4 channel) and the other during the S 2 -S 3 and S 3 -S 0 transitions (Cl1 channel). The O1 substrate water delivery channel has a short, highly conserved section up to the single point water valve that likely opens and closes water access at the right moments in the mechanism via allosteric control 36 , 37 . After this bottleneck, the only requirement is that water can freely reach this point and thus it can vary dramatically between species without functional consequences. The design of the two proton channels is very different: the O4 channel has an extended line of well-ordered water molecules who’s H-bonding pattern has been proposed to open/close the proton egress pathway 42 , 45 . By contrast, the Cl1 channel is less restricted, and the proton egress pathway involves several conserved charged amino acids and is regulated by proton gate residues that likely open by electrostatic control 10 . We thus speculate that the Cl1 proton exit path can only operate in the higher S states when the positive extra charge is present as in the S 2 and S 3 states. In these states, the Cl1 channel is more efficient for proton release than the O4 channel, while in the neutral S 0 state, the O4 channel can be used for proton egress. The present advancement in the resolution of plant PS II has allowed a level of comparison to cyanobacterial PS II that was previously impossible. Through these comparisons, it is possible to determine which features of the protein and water network of PS II are essential, providing insight into design principles of photosynthesis and enzyme catalysis as a whole. Methods Photosystem II extraction, purification and activity assay Arabidopsis thaliana (Columbia-0) plants were grown for 8 weeks at constant temperature (20 o C) and humidity (70%), and day/night periods of 8 h light/16 h dark. The growth of these plants complied with the local and national regulations and the permission was given from Jordbruksverket-Växtkontrollenheten (Dnr 4.6.20–6365/15). The leaves of the plants were harvested after 16 hours of dark, and BBY membranes 46 were extracted from the leaves as described in Chen et al., 2019 47 . BBY membranes were washed with wash buffer (25 mM MES pH 6.3, 10 mM NaCl, 5 mM CaCl 2 ) and resuspended in wash buffer at a concentration of 1 mg mL − 1 Chlorophyll. BBY membranes were then diluted to 0.5 mg.mL − 1 with 0.6% (w/v) n-dodecyl-β-maltoside (β-DM) in wash buffer, giving a final concentration of 0.3% β-DM and left with slow spinning for 1 min. Insoluble material was pelleted by centrifuging at 16,000 x g for 5 min, before transferring the supernatant and spinning again at 20,000 x g for 10 min. The supernatant was loaded onto the top of sucrose gradients which had been formed by the freeze-thawing method (25 mM MES pH 6.3, 0.5 M sucrose, 1 M betaine, 10 mM NaCl, 5 mM CaCl 2 , 0.01% (w/v) β-DM (Anatrace, U.S.A). Sucrose gradients were centrifuged at 141,000 x g for 18 h, and the resulting bands were separated. Oxygen evolution measurements were made on the separated bands using a Clark-type electrode (Hansatech). The bands were measured at a chlorophyll concentration of 10 µg mL − 1 of chlorophyll with 0.2 mM PPBQ (Phenyl-p-benzoquinone) and of 1 mM K 3 FeCN 6 added as electron acceptors using saturating light (Supplementary Fig. 9). Sample preparation and electron microscopy C 2 S 2 M 2 and C 2 S 2 complexes were concentrated using 100 kDa molecular weight cut-off spin filters (Amicon) and then washed several times with pre-freezing buffer (50 mM MES pH 6.3, 20 mM CaCl 2 , 5 mM MgCl 2 , 0.6 M Betaine, 0.03% (w/v) β-DM) before finally concentrating to a final concentration of 1.6 mg Chl mL − 1 . 4 µL of sample was loaded onto glow-discharged (30s at 50 mA) Quantifoil grids R 1.2/1.3 Cu 300 (Quantifoil Micro Tools) in dim room light. The grids were then plunge frozen in liquid ethane using an FEI Vitrobot MkIV (Thermo Fisher Scientific) at 100% humidity, 4 o C, and a blot force of -5, a wait time of 1 s and a blotting time of 5 s. Automated data collection was performed using EPU software on a Titan Krios G2 transmission electron microscope operating at 300 kEV (Thermo Fisher Scientific) at the Umeå Core Facility for Electron Microscopy, a node of the Swedish National Cryo-EM facility. The Titan Krios was equipped with Falcon 4 direct electron detectors and a Selectris energy filter. In total 16,450 movies were collected, which each contained 40 frames. The movies were collected at a pixel size of 0.704 Å, a total dose of 40 electrons/Å 2 and defocus values that ranged from − 0.8 to -2.0. Data processing The dataset was processed using cryoSPARC software. Motion and contrast transfer function corrections were performed before template free picking was used to identify particles. 3,054,062 particles were initially identified, before several rounds of 2D classification reduced the number of “good” particles to 322,522. Ab initio 3D reconstruction was used to generate 6 different 3D volumes, which were then refined using heterogeneous refinement. The particles used to generate the best-looking class went through additional filtering using 2D classification and multi-class ab initio 3D reconstruction until a final volume was generated using 72,301 particles and refined using homogeneous refinement and non-uniform refinement with imposed C2 symmetry. The final volume had an estimated global resolution of 2.44 Å, using the gold standard FSC (Supplementary Fig. 10). Finally, the map was sharpened, using Phenix to perform local sharpening with the Autosharpen function. Model building The model for spinach photosystem II (3JCU) was used as a starting point and manually fitted into the map using UCSF ChimeraX software (version 1.7) 48 . COOT (version 0.9.8.92) 49 was used to perform the relevant mutations to change the Spinacia oleracea protein sequences to the corresponding Arabidopsis thaliana sequences. COOT was then used to refine the model to fit into the sharpened map. Several rounds of automatic fitting and manual fitting/checking were performed to optimize the fitting of the model into the map densities. Final refinement was performed using the Real-space refinement function in Phenix 50 , with manual checking in COOT to confirm or correct the automatic refinement. All structures in the figures were prepared with ChimeraX or Pymol. Details of the data collection and model building are shown in Supplemental tables 1–3. Water channel calculations The channels connecting the Manganese cluster to the exterior of the protein were calculated using CAVER software 51 (version 3.0.3), which is a plugin for PyMOL 52 . For most of the channels, standard settings were used: probe radius 0.9, shell depth 4, shell radium 3, clustering threshold 3.5. For the Cl1 channel, it was necessary to reduce the probe radius to 0.6. B-factor analysis The structure was refined using REFMAC5 53 , implemented in Servalcat, with the two unsharpened half-maps and the required mask file. Figures related to the analysis were prepared using PyMOL 52 . Declarations Acknowledgements The cryo-electron microscopy data was collected at Umeå Core Facility for Electron Microscopy, a node of the SciLifeLab National Cryo-EM Facility, funded by the Knut and Alice Wallenberg Foundation, the Erling-Persson Foundation,the Kempe Foundation, SciLifeLab, Stockholm University, and Umeå University. This work was supported by Vetenskapsrådet grant 2020-03809 (J.M.); Carl Tryggers Foundation (CTS 19.324) (W.P.S.); Kempe (JCK-2030 2021-2023) (W.P.S. and J.M.); German Research Foundation (DFG) grant through the Collaborative Research Center SFB1078 (Humboldt Universität zu Berlin, grant no.TP A5); SFB 1507 “Membrane-associated protein assemblies, machineries, and supercomplexes” (R.H.) and the Lawski foundation (date 240612) (A.G. and J.F.). 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In Silico and biochemical analysis of Physcomitrella patens photosynthetic antenna: Identification of subunits which evolved upon land adaptation. PLoS ONE , 3(4), e2033. Cardona, T., Sánchez-Baracaldo, P., Rutherford, A. W., Larkum, A. W. (2018). Early Archean origin of Photosystem II. Geobiology, 17(2), 127–150. Retegan, M. and Pantazis, D. A. (2017). Differences in the active site of water oxidation among photosynthetic organisms. J. Am. Chem. Soc., 139(41), 14340–14343. Bricker, T.M., Roose, J.L., Fagerlund, R.D., Frankel, L.K. Eaton-Rye, J.J. (2012) The extrinsic proteins of Photosystem II. BBA - Bioenergetics, 1817(1), 121–142. Umena, Y., Kawakami, K., Shen, J., Kamiya, N. (2011). Crystal structure of oxygen-evolving Photosystem II at 1.9 angstrom resolution. Nature, 473(7345), 55–60. Kern, J., Chatterjee, R., Young, I. D., Fuller, F. D., Lassalle, L., Ibrahim, M.,Gul, S., Fransson, T., Brewster, A. S., Alonso-Mori, R., Hussein, R., Zhang, M., Douthit,L., De Lichtenberg, C., Cheah, M. H., Shevela, D., Wersig, J., Seuffert, I., Sokaras,D., … Yachandra, V. K. (2018). Structures of the intermediates of Kok’s photosynthetic water oxidation clock. Nature , 563 (7731), 421–425. Suga, M., Akita, F., Hirata, K., Ueno, G., Murakami, H., Nakajima, Y., Shimizu, T., Yamashita, K., Yamamoto, M., Ago, H., Shen, J. (2015). Native structure of photosystem II at 1.95 Å resolution viewed by femtosecond X-ray pulses. Nature, 517 (7532), 99–103. Bhowmick, A., Hussein, R., Bogacz, I., Simon, P., Ibrahim, M., Chatterjee, R., Doyle,M.D, Cheah, M-H., Fransson, T., Chernev, P., Kim, I-S, Makita, H., Dasgupta, M., Kaminsky,C.J., Zhang, M., Gätcke, J., Haupt, S., Nangca, I.I., Keable, S.M.,… Yachandra, V.K.(2023) Structural evidence for intermediates during O 2 formation in photosystem II. Nature 617, 629–636. Kato, K., Miyazaki, N., Hamaguchi, T., Nakajima, Y., Akita, F., Yonekura, K., & Shen, J. (2021). High-resolution cryo-EM structure of photosystem II reveals damage from high-dose electron beams. Commun. Biol., 4(1), 1–11. Gisriel CJ, Wang J, Liu J, Flesher DA, Reiss KM, Huang HL, Yang KR, Armstrong WH, Gunner MR, Batista VS, Debus RJ, Brudvig GW.(2022) High-resolution cryo-electron microscopy structure of photosystem II from the mesophilic cyanobacterium, Synechocystis sp. PCC 6803. PNAS, 119(1), 1–10. Hussein, R., Graça, A., Forsman, J., Aydin, A. O., Hall, M., Gaetcke, J., Chernev, P., Wendler, P., Dobbek, H., Messinger, J., Zouni, A., Schröder, W. P. (2024). Cryo–electron microscopy reveals hydrogen positions and water networks in photosystem II. Science, 384(6702), 1349–1355. Wei, X., Su, X., Cao, P., Liu, X., Chang, W., Li, M., Zhang, X., Liu, Z. (2016). Structure of spinach photosystem II–LHCII supercomplex at 3.2 Å resolution. Nature, 534(7605), 69–74. Su X, Ma J, Wei X, Cao P, Zhu D, Chang W, Liu Z, Zhang X, Li M. (2017) Structure and assembly mechanism of plant C 2 S 2 M 2 -type PSII-LHCII supercomplex. Science, 357(6353), 815–820. Opatíková, M., Semchonok, D. A., Kopečný, D., Ilík, P., Pospíšil, P., Ilíková, I., Roudnický, P., Zeljković, S. Ć., Tarkowski, P., Kyrilis, F. L., Hamdi, F., Kastritis, P. L., Kouřil, R. (2023). Cryo-EM structure of a plant photosystem II supercomplex with light-harvesting protein Lhcb8 and α-tocopherol. Nat. Plants, 9(8), 1359–1369. Graça, A. T., Hall, M., Persson, K., Schröder, W. P. (2021). High-resolution model of arabidopsis Photosystem II reveals the structural consequences of digitonin-extraction. Scientific Reports , 11(1). Sirohiwal, A., Pantazis, D.A. (2022) Functional water networks in fully hydrated Photosystem II. J. Am. Chem. Soc., 144(48), 22035–22050. Ishikita, H. and Knapp, E. (2005). Control of quinone redox potentials in Photosystem II: electron transfer and Photoprotection. J. Am. Chem. Soc., 127(42), 14714–14720. Saito, K., Rutherford, A. W., Ishikita, H. (2012). Mechanism of proton-coupled quinone reduction in Photosystem II. PNAS, 110(3), 954–959. Sugo, Y. and Ishikita, H. (2022). Proton-mediated photoprotection mechanism in photosystem II. Front. Plant Sci., 13. Sugo, Y., Saito, K., Ishikia, H. (2022) Conformational changes and H-bond rearrangements during quinone release in Photosystem II Biochemistry, 61, 1836–1843. Li, H., Nakajima, Y., Nango, E., Owada, S., Yamada, D., Hashimoto, K., Luo, F., Tanaka,R., Akita, F., Kato, K., Kang, J., Saitoh, Y., Kishi, S., Yu, H., Matsubara, N., Fujii,H., Sugahara, M., Suzuki, M., Masuda, T., Kimura, T.,… Shen, J-R (2024) Oxygen-evolving photosystem II structures during S 1 -S 2 -S 3 transitions Nature 626, 670–677. Suga, M., Akita, F., Yamashita, K., Nakajima, Y., Ueno, G., Li, H., Yamane, T., Hirata,K., Umena, Y., Yonekura, S., Yu, L., Murakami, H., Nomura, T., Kimura, T., Kubo, M.,Baba, S., Kumasaka, T., Tono, K., Yabashi, M., … Shen, J. (2019). An oxyl/oxo mechanism for oxygen-oxygen coupling in PSII revealed by an X-ray free-electron laser. Science , 366(6463), 334–338. Shimada, Y., Kitajima-Ihara, T., Nagao, R., Noguchi, T. (2020) Role of the O4 channel in photosynthetic water oxidation as revealed by fourier transform infrared difference and time-resolved infrared analysis of the D1-S169A mutant. J. Phys. Chem. B, 124 (8), 1470–1480. Vassiliev, S., Zaraiskaya, T., Bruce, D. (2012). Exploring the energetics of water permeation in photosystem II by multiple steered molecular dynamics simulations. BBA - Bioenergetics, 1817 (9), 1671–1678. Linke, K., Ho, F. M. (2014). Water in Photosystem II: Structural, functional and mechanistic considerations. BBA - Bioenergetics, 1837 (1), 14–32. De Lichtenberg, C., Kim, C. J., Chernev, P., Debus, R. J., Messinger, J. (2021). The exchange of the fast substrate water in the S 2 state of photosystem II is limited by diffusion of bulk water through channels – implications for the water oxidation mechanism. Chem. Sci., 12 (38), 12763–12775. Kaur, D., Reiss, K., Wang, J., Batista, V.S., Brudvig, G.W., Gunner, M.R. (2024) Occupancy Analysis of Water Molecules inside Channels within 25 Å Radius of the Oxygen-Evolving Center of Photosystem II in Molecular Dynamics Simulations J. Phys. Chem. B, 128(10) 2236–2248. Sakashita, N., Watanabe, H. C., Ikeda, T., Ishikita, H. (2017) Structurally conserved channels in cyanobacterial and plant photosystem II. Photosynth. Res., 133(1–3) 75–85. Grinzato, A., Albanese, P., Marotta, R., Swuec, P., Saracco, G., Bolognesi, M., Zanotti, G., Pagliano, C. (2020) High-Light versus Low-Light: Effects on Paired Photosystem II Supercomplex Structural Rearrangement in Pea Plants. Int. J. Mol. Sci., 21 , 8643. Shimada, Y., Kitajima-Ihara, T., Nagao, R., Noguchi, T. (2020) Role of the O4 channel in photosynthetic water oxidation as revealed by fourier transform infrared difference and time-resolved infrared analysis of the D1-S169A mutant J. Phys. Chem. B 124, 1470–1480. Berthold, D. A., Babcock, G. T., Yocum, C. F. (1981) A highly resolved, oxygen evolving photosystem II preparation from Spinach thylakoid membranes. Fed. Eur. Biochem. Soc. Lett. 134, 231–234. Chen, Y.E., Yuan, S., Lezhneva, L., Meurer, J., Schwenkert, S., Mamedov, F., Schröder, W.P. (2019) The Low Molecular Mass Photosystem II Protein PsbTn Is Important for Light Acclimation. Plant Physiol. 179(4), 1739–1753. Meng, E.C., Goddard, T.D., Pettersen, E.F., Couch, G.S., Pearson, Z.J., Morris, J.H., Ferrin, T.E. (2023) UCSF ChimeraX: Tools for structure building and analysis Protein Sci. 32(11) e4792. Emsley, P., Lohkamp, B., Scott, W. G., Cowtan, K. (2010) Features and development of Coot. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 486–501. Afonine, V., Poon, B. K., Read, R. J., Sobolev, O. V., Terwilliger, T. C., Urzhumtsev, A., Adams, P. D. (2018) Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D Struct. Biol. 74, 531–544 Chovancová,E., Pavelka, A., Beneš, P., Strnad, O., Brezovský, J., Kozlíková, B., Gora, A., Šustr, V., Klvaňa, M., Medek, P., Biedermannová, L., Sochor, J., Damborský, J (2012) CAVER 3.0: A Tool for the Analysis of Transport Pathways in Dynamic Protein Structures, PLoS Comput. Biol. 8(10), e1002708. Schrödinger, L. (2015) The PyMOL molecular graphics system. Verson¸ 3.1.0 Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan, R. M., Krissinel, E. B., Leslie, A. G. W., McCoy, A., McNicholas, S. J., Murshudov, G. N., Pannu, N. S., Potterton, E. A., Powell, H. R., Read, R. J., Vagin, A., Wilson, K. S. (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242. Additional Declarations There is NO Competing Interest. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5656066","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":399502465,"identity":"e4437aee-a202-481f-a890-9c99ec0d6b8c","order_by":0,"name":"Johannes Messinger","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIie2PMUvEMBiGv1LolNBNit/gX8hR6HJH81caAp1yqItzp9xSdb3+CydxrGQNt8u5iHB7OZCCgrbHCaJEcRPMMyTkJQ/v9wF4PH+QoCIfn2w8wvbXSlT80EO+JuxbIVyc325Pb2YcDozp+pP7/BLt0+GdBh67BqtXEhtbigrLEgnbyOZifo1zDaKpHMpSMaTaFIAqC4EZySzdKQVrnUr6TPUrBzzedv2gcEs2o8K5W8mGljaoUEFCmMkZIdGoBFfOXWw2pVoKjWU27GKKxEbpVK0SsXS0TBZ1uqY65zHKx65/MTyuw4e1Opvx2LH+5D2P9rfYB4ljLICjzwF3fvV4PJ5/yxsWUVMYME4PiwAAAABJRU5ErkJggg==","orcid":"","institution":"Umeå University","correspondingAuthor":true,"prefix":"","firstName":"Johannes","middleName":"","lastName":"Messinger","suffix":""},{"id":399502466,"identity":"90db727f-7db2-4d3f-9bac-7ea228611b19","order_by":1,"name":"Jack Forsman","email":"","orcid":"","institution":"Uppsala University","correspondingAuthor":false,"prefix":"","firstName":"Jack","middleName":"","lastName":"Forsman","suffix":""},{"id":399502467,"identity":"e14a6c5d-e4a3-4847-b8e3-765788092173","order_by":2,"name":"André Graca","email":"","orcid":"","institution":"Umeå University","correspondingAuthor":false,"prefix":"","firstName":"André","middleName":"","lastName":"Graca","suffix":""},{"id":399502468,"identity":"2e993ce7-5465-410c-9019-920c54d6cce0","order_by":3,"name":"Abuzer Aydin","email":"","orcid":"","institution":"Uppsala University","correspondingAuthor":false,"prefix":"","firstName":"Abuzer","middleName":"","lastName":"Aydin","suffix":""},{"id":399502469,"identity":"f959b8e9-29ff-42bc-85ad-f2a4bf959c06","order_by":4,"name":"Michael Hall","email":"","orcid":"","institution":"Umea University","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Hall","suffix":""},{"id":399502470,"identity":"dde9565e-9397-4bcf-a30f-97cd778c5867","order_by":5,"name":"Rana Hussein","email":"","orcid":"","institution":"Humboldt University","correspondingAuthor":false,"prefix":"","firstName":"Rana","middleName":"","lastName":"Hussein","suffix":""},{"id":399502471,"identity":"07228a63-bf96-4e61-9946-4f12a1116537","order_by":6,"name":"Wolfgang Schröder","email":"","orcid":"https://orcid.org/0000-0002-9492-5113","institution":"Umeå University","correspondingAuthor":false,"prefix":"","firstName":"Wolfgang","middleName":"","lastName":"Schröder","suffix":""}],"badges":[],"createdAt":"2024-12-16 18:11:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5656066/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5656066/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73361993,"identity":"99d02d89-6fb2-4942-b05e-0864938f1713","added_by":"auto","created_at":"2025-01-09 08:54:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":886890,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Side view of photosystem II from Arabidopsis thaliana with essential cofactors. The Arabidopsis model shown in white transparent cartoons with the Coulomb potential map in transparent blue. Important cofactors are highlighted in the structure: Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster (red, purple and yellow balls), P680 pigments (chlorophyll a molecules in green and pheophytin molecules in blue), Q\u003csub\u003eA\u003c/sub\u003e and Q\u003csub\u003eB\u003c/sub\u003e molecules (yellow), non-heme iron (orange sphere), redox active tyrosines Tyr\u003csub\u003eZ\u003c/sub\u003e and Tyr\u003csub\u003eD\u003c/sub\u003e (cyan). Black arrows show the direction of electron transfer during water splitting. \u003cstrong\u003e(B) \u003c/strong\u003eTop view of photosystem II from Arabidopsis thaliana. The Coulomb potential map is shown in transparent blue with model built into the map (white transparent cartoons). Chlorophyll molecules in the model are shown in light green (Chlorophyll a) or dark green (Chlorophyll b). The monomer:monomer interface is indicated with a black dashed line and labelled. For one of the monomers the LHC II strongly bound trimer (s-LHC II), CP26 and CP29 subunits are circled and labelled.\u003cstrong\u003e \u003c/strong\u003eSide view \u003cstrong\u003e(C)\u003c/strong\u003e and top view \u003cstrong\u003e(D)\u003c/strong\u003e of the map of photosystem II coloured to show the local resolution. Scale bar indicating the local resolution is shown in the bottom right.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5656066/v1/cf1c5155652fe86e4c6b945e.png"},{"id":73362564,"identity":"a5a30359-1fd5-4a0c-bd4c-2052ecc6eb5e","added_by":"auto","created_at":"2025-01-09 09:02:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":781702,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e The environment of the previously unmodelled β-carotene is shown for one of the monomers. The β-carotene is shown in red. The electrostatic density from the local sharpened Coulomb potential map within 1.5 Å of the β-carotene is shown as a mesh to demonstrate the accuracy of the fitting of the new β-carotene. The contour level of the locally sharpened map is 1.38 RMSD. The nearest subunits to the new β-carotene molecule are D1 (yellow), PsbI (green) and PsbO (cyan). The nearest chlorophyll molecule (Chla:A410) is shown in lime green. The closest interactions between this new β-carotene and the surrounding proteins are: 4.1 Å to D1:Val102, 3.9 Å to PsbI:Phe15, 3.2 Å to PsbI:Met1, 3.9 Å to PsbO:Lys160, 5.2 Å to Chla:A410. \u003cstrong\u003e(B)\u003c/strong\u003e Overlay view of a cluster of β-carotene molecules found in at the monomer:monomer interface from a range of different structures. All β-carotene molecules in this area are shown from Arabidopsis (red, current structure), T. vestitus (blue, 9EVX), spinach (pink, 3JCU), pea (green, 5NXL) and spruce (yellow, 8C29). The structure of the surrounding area is shown as white cartoons \u003cstrong\u003e(C) \u003c/strong\u003eThe location of the newly identified cofactors within the C\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e-PS II complex. Digalactosyl diacylglycerol (DGD) and sulfoquinovosyl diacylglycerol (SQD) molecules are shown in blue and green, respectively. The β-carotene is shown in red. The Coulomb potential map of PSII (top view) from Arabidopsis is shown in the background in pale blue with the Arabidopsis model is shown as a white cartoon.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5656066/v1/49ff10c172c8dd1b1dbf13fb.png"},{"id":73361996,"identity":"9a73ba16-d634-4950-82a6-dc9b5cce223e","added_by":"auto","created_at":"2025-01-09 08:54:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":422995,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Comparison of the water positions within 20 Å of\u003cem\u003e \u003c/em\u003ethe bicarbonate ligand to the non-heme iron in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (current structure) or \u003cem\u003eT\u003c/em\u003e. \u003cem\u003evestitus\u003c/em\u003e (9EVX). Waters from Arabidopsis are shown as green spheres, waters from \u003cem\u003eT\u003c/em\u003e. \u003cem\u003evestitus\u003c/em\u003e are shown as grey spheres. For clarity, some water molecules and residues have been omitted. The locally sharpened Coulomb potential map at a contour level of 1.29 RMSD is shown as a mesh around the Arabidopsis waters. Selected D1 and D2 residues from Arabidopsis are shown in white. The D1:His215 and D1:Tyr246 residues shown in green with blue and red sections indicating nitrogen or oxygen atoms, respectively. Q\u003csub\u003eA\u003c/sub\u003e and Q\u003csub\u003eB\u003c/sub\u003e quinones are shown in yellow. The bicarbonate ligand is shown in white, with red sections representing oxygen atoms. Water 63 (W63) plays a role in re-protonation of D1:His215. The distances between W63 and D1:His215, D1:Tyr246 and the bicarbonate are shown as dashed black lines, these distances are: 4.0 Å, 2.3 Å and 3.4 Å, respectively.\u003cstrong\u003e (B)\u003c/strong\u003e Comparison of the protein structure around the QB binding pocket in \u003cem\u003eArabidopsis thaliana \u003c/em\u003e(green, current structure) and \u003cem\u003eT. vestitus\u003c/em\u003e (grey, 9EVX). The whole PsbA (D1) subunits have been aligned using the ChimeraX tool “Matchmaker”. The Q\u003csub\u003eB\u003c/sub\u003e, D1:His252, D1:Ser264 and D1:Ile.248 residues are shown as sticks. Red arrows indicate the movement of the D1:247-267 region. A double headed red arrow indicates the different side chain orientation of the D1:Ile248 residue in the two structures. Waters modelled within the Q\u003csub\u003eB\u003c/sub\u003e binding pocket are circled with red dotted lines. These waters are not found in the \u003cem\u003eT. vestitus\u003c/em\u003e (9EVX) structure. \u003cstrong\u003e(C)\u003c/strong\u003e The acceptor side of PS II from the current structure. The structure is coloured to indicate the b-factor. Light green to red colour gradient and a wider tube indicate regions with higher B-factor. The scale bar is shown below the panel.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5656066/v1/30b937ea4a04be9d13aefc8a.png"},{"id":73362005,"identity":"f265d377-3bc9-4b57-ba2f-d4ab74881f63","added_by":"auto","created_at":"2025-01-09 08:54:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":502694,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Comparison of the water positions within 9 Å of the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster in Arabidopsis thaliana (current structure) or T. vestitus (9EVX). Waters from Arabidopsis are shown as green spheres, waters from T. vestitus are shown as grey spheres. The Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster is shown as a cluster of red, purple or yellow spheres, representing O, Mn or Ca ions, respectively. Black lines indicate bonds connecting specific water molecules to the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster. These bound waters are labelled as W1, W2, W3 and W4. The W200 water is also labelled and circled with a red dotted line. This water has no corresponding water in the T. vestitus structure (9EVX). The water wheel is indicated with lines connecting the constituent waters. The O1, O4 and Cl1 channels which lead to the exterior of the protein in the current Arabidopsis structure are shown in red, blue and green, respectively (Channels calculated using the CAVER plugin for Pymol). The D1:87 residue differs between plants and cyanobacteria. In plants like Arabidopsis thaliana, the D1:87 residue is an alanine and it its side chain is shown with a green stick. In cyanobacteria, here T. vestitus, the D1:87 residue is an asparagine and its side chain is shown with grey sticks (red and blue sections indicate oxygen and nitrogen atoms, respectively). \u003cstrong\u003e(B) \u003c/strong\u003eElectron density from the locally sharpened Coulomb density map at a contour level of 1.29 RMSD is shown as a mesh around the Arabidopsis water molecules. The W1-W4 waters were excluded for clarity; to see the Coulomb density for the W1-W4 waters and the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster see Sup. Fig. 1. \u003cstrong\u003e(C) \u003c/strong\u003eElectron density from the locally sharpened Coulomb potential map at a contour level of 0.49 RMSD is shown as a mesh around the Arabidopsis water. Specifically, at this resolution all waters in the water wheel are surrounded by Coulomb density.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5656066/v1/79677e3a633f172ba487ed77.png"},{"id":73362003,"identity":"ec06d3e3-9654-4f9f-8335-6753adbf1aa6","added_by":"auto","created_at":"2025-01-09 08:54:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":309592,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e O1 channels connecting the O1 atom of the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster to the exterior of the protein. Channels calculated for the current Arabidopsis structure are shown in red, channels calculated for the T. vestitus structure (9EVX) are shown in grey. The various branches of the O1 channels are labelled with a letter (A-C). The Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster is shown as a cluster of red, purple or yellow spheres, representing O, Mn or Ca atoms, respectively. All branches of the O1 channels in both Arabidopsis and T. vestitus pass through a common bottleneck. The residues which form this bottleneck are shown in green with blue and red sections indicating nitrogen or oxygen atoms, respectively. For both species these bottleneck residues are: D1:Glu329, D1:Pro340, D1:Asp342, and CP43:Val410. Only one water molecule could be modelled inside the O1 channels for Arabidopsis and was found in the bottleneck region (green sphere). 19 waters were modelled in the O1 channels of T. vestitus (9EVX), these are shown as grey spheres. \u003cstrong\u003e(B)\u003c/strong\u003e Potential hydrogen bonding interactions between the water modelled at the bottleneck region of the O1 channel and the surrounding protein structure. A water in this position is found in Arabidopsis (current structure, green sphere), T. vestitus (9EVX, grey sphere) and Pea (5XNL, cyan sphere). Potential hydrogen bonds are shown as black dashed lines, between the water and the D1:E329, D1:P340 and D1:D342 residues, corresponding to distances of 2.7 Å, 3.5 Å and 3.0 Å, respectively. The mesh around the bottleneck water represents the Coulomb density from the locally sharpened Coulomb potential map at a contour level of 1.29 RMSD. The surrounding residues are shown in white with red and blue sections indicating oxygen and nitrogen atoms, respectively.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5656066/v1/f1e513eb95b138bb7fad8627.png"},{"id":73362012,"identity":"3fe166ab-f8bd-4ef5-b2b3-2f14b2737665","added_by":"auto","created_at":"2025-01-09 08:54:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":296750,"visible":true,"origin":"","legend":"\u003cp\u003eChannels connecting the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster to the protein surface. For the current Arabidopsis structure, the Cl channel is shown in green and the O4 channel in blue, while the corresponding channels of T. vestitus (9EVX) are shown in grey. The Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster is shown as a cluster of red, purple or yellow spheres, representing O, Mn and Ca ions, respectively. \u003cstrong\u003e(A)\u003c/strong\u003e The Cl1 channel. The residues which form the bottleneck region in the Cl1 channel are shown in green for Arabidopsis and in grey for T. vestitus, with blue and red sections indicating nitrogen or oxygen atoms, respectively. These bottleneck residues are: D1:Glu65, D1:Pro66 and D2:Glu312. The D1:Asp61 residue is also shown, as the D1:Asp61, D2:Glu312 and D1:Glu65 residues are important for proton transfer. Water molecules identified in the present Arabidopsis model are shown as green spheres, waters from the T. vestitus model (9EVX) are shown as grey spheres. The nearby chloride atom Cl1 is shown in light green. \u003cstrong\u003e(B)\u003c/strong\u003eThe O4 channel. The D1:Asn338, and CP43:Gly333; CP43:Pro334 residues form the bottleneck region and are shown in green. Water molecules found in the present Arabidopsis model are shown as green spheres, waters from T. vestitus (9EVX) are shown as grey spheres.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5656066/v1/52217f4045a18d1eaef84f4d.png"},{"id":77347716,"identity":"fca90e2c-f004-45f4-9ad3-ee5ba8eaa2d6","added_by":"auto","created_at":"2025-02-27 15:59:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4000487,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5656066/v1/4e1eaf5a-3c28-48e0-9e27-d759a9280a39.pdf"},{"id":73361999,"identity":"93da5e18-ba53-4484-abc7-d77bf0d4595f","added_by":"auto","created_at":"2025-01-09 08:54:52","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4112098,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"ArabidopsisSupplementalmaterial1.1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5656066/v1/c96bd2511d528b11ce81bf95.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Structure of the intact C2S2-type Photosystem II-LHCII supercomplex from Arabidopsis thaliana at 2.44 Å","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePhotosystem II (PS II) is a large protein complex located in the thylakoid membranes of oxygen-evolving photosynthetic organisms. PS II absorbs light energy and uses it to perform charge separation reactions that drive the oxidation of water and the reduction of plastoquinone\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. This reaction results in the production of molecular oxygen, making it essential to life on Earth.\u003c/p\u003e \u003cp\u003eThe D1 and D2 subunits in the PS II core coordinate the cofactors required to perform water splitting. This includes the chlorophyll (P680) and pheophytin (Pheo) molecules, between which the primary charge separation occurs; and the manganese, calcium, oxygen cluster (Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster) that oxidizes water (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA)\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The energy required to perform water splitting is primarily captured, from sunlight, by peripheral antenna proteins and funneled toward the core antenna subunits CP43 and CP47, before being passed to P680\u003csup\u003e3\u003c/sup\u003e. In addition, there are several small transmembrane proteins that support the function of PS II in various ways, and the extrinsic subunits which stabilize the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster is sequentially oxidized by successive charge separations between P680 and Pheo, facilitating the binding, de-protonation and oxidation of the two substrate water molecules. This process, known as the Kok-cycle\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, transitions the cluster from its most reduced state, S\u003csub\u003e0,\u003c/sub\u003e through the intermediate states S\u003csub\u003e1\u003c/sub\u003e, S\u003csub\u003e2\u003c/sub\u003e, S\u003csub\u003e3,\u003c/sub\u003e and the transient S\u003csub\u003e4\u003c/sub\u003e state, leading to the formation of molecular oxygen during the S\u003csub\u003e4\u003c/sub\u003e\u0026loz;S\u003csub\u003e0\u003c/sub\u003e transition\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster is connected to the water phase of the lumen via channels\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. These channels allow water to access the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster and protons to egress. Recently, it has been shown that a specific arrangement of the water network around the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster is required for efficient water insertion and proton release during the S\u003csub\u003e2\u003c/sub\u003e\u0026loz;S\u003csub\u003e3\u003c/sub\u003e and the S\u003csub\u003e3\u003c/sub\u003e\u0026loz;S\u003csub\u003e0\u003c/sub\u003e transitions\u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Consequently, the water network around the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster and the proton gate must be carefully regulated to prioritize the specific conformations required for these events to occur.\u003c/p\u003e \u003cp\u003eAcross all photosynthetic organisms, the structures of the core subunits are highly conserved (D1, D2, CP43 and CP47)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, a notable difference exists near the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster at position 87 of the D1 protein, where an alanine residue is found in plants and an asparagine residue is present in the majority of cyanobacteria, affecting the water and H-bonding networks in ways that remain to be explored\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Additionally, different photosynthetic species exhibit significant variation in the small transmembrane and extrinsic proteins\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, which may alter the channels to the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster and thereby, these networks\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe ability of PS II from thermophilic cyanobacteria to readily form crystals allowed several high-resolution x-ray diffraction structures of PS II to be collected from these organisms (\u003cem\u003eThermosynechococcus vestitus\u003c/em\u003e or \u003cem\u003eThermosynechococcus vulcanus\u003c/em\u003e) with resolutions up to 1.89 \u0026Aring;\u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Recent improvements to cryo-electron microscopy (cryo-EM) have also resulted in several high-resolution structures of PS II from cyanobacteria with resolutions up to 1.71 \u0026Aring;\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. However, the structure of PS II from land plants has been more difficult to obtain. Low/medium-resolution structures of PS II are available from \u003cem\u003eSpinacia oleracea\u003c/em\u003e (spinach) (3JCU)\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003ePisum sativum\u003c/em\u003e (pea) (5XNL)\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003ePicea abies\u003c/em\u003e (spruce) (8C29)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, with resolutions of 3.2 \u0026Aring;, 2.7 \u0026Aring; and 2.8 \u0026Aring;, respectively. Additionally, our research group has previously published a structure of PS II from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (7OUI)\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e at a resolution of 2.79 \u0026Aring;. However, this structure lacked the PsbP, PsbQ and PsbJ subunits, and the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster. Here, we present a fully intact C\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e-PS II:LHC II supercomplex from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e at a resolution of 2.44 \u0026Aring;.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eC\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e-type Photosystem II map and model\u003c/h2\u003e \u003cp\u003eThe Coulomb potential map of the C\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e-type PS II was generated from 72,301 particles with enforced C2 symmetry and reached a global resolution of 2.44 \u0026Aring;, ranging from 2 \u0026Aring; resolution in the core to 3.5 \u0026Aring; resolution in the flexible peripheral regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-D).\u003c/p\u003e \u003cp\u003eThe map showed clear densities for all the PS II core subunits and all extrinsic subunits, as well as for all metal ions and water ligands of the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster (Supplementary Fig.\u0026nbsp;1A). The Mn-Mn and Mn-Ca distances in the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster are elongated in comparison with XFEL structures\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, suggesting partial reduction of the Mn ions during data collection (micrographs collected at 40 e\u003csup\u003e\u0026minus;\u003c/sup\u003e/\u0026Aring;\u003csup\u003e\u0026minus;2\u003c/sup\u003e). In contrast, the proteins appear practically unchanged; the disulfide bond in PsbO protein, which breaks at high electron dose (83 e\u003csup\u003e\u0026minus;\u003c/sup\u003e/\u0026Aring;\u003csup\u003e\u0026minus;2\u003c/sup\u003e)\u003csup\u003e24\u003c/sup\u003e, is intact in our structure (Supplementary Fig.\u0026nbsp;1B). The strongly bound LHC II trimers were successfully modeled (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB); however, the map showed no density for the mediumly-bound LHC II trimers in either the 3D structure or the 2D classes. For subunits with multiple similar isoforms, the model displays the best fitting isoform (Supplementary Fig.\u0026nbsp;2), although the map may contain contributions from multiple different isoforms.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCofactors\u003c/h3\u003e\n\u003cp\u003eOur map showed a clear density at the monomer:monomer interface which we modelled as a β-carotene (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This β-carotene molecule (named PsbI:BCR34) was found close to the PsbI subunit in both monomers. Other PS II cryo-EM maps either show no feature at this position (spinach, 3JCU), or the electrostatic potential is filled with a steric acid (\u003cem\u003eT. vestitus\u003c/em\u003e, 9EVX), a lineolenic acid (spruce, 8C29), or a distearoyl-monogalactosyl-diglyceride (LMG) (pea, 5XNL). Our new β-carotene model fits reasonably well into the electron/charge densities from the other maps, suggesting that this cofactor may also be present in the other species (Supplementary Fig.\u0026nbsp;3). At the closest point PsbI:BCR 34 is 5.2 \u0026Aring; from chlorophyll \u003cem\u003ea\u003c/em\u003e 410 in the D1 subunit, (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). It is therefore feasible that this β-carotene may interact with this chlorophyll as either an antenna or a quencher. All other β-carotene molecules in our structure can also be found in the \u003cem\u003eT. vestitus\u003c/em\u003e (9EVX), pea (5XNL), spinach (3JCU) or spruce (8C29) structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur new map also allowed the lipids digalactosyl diacylglycerol (DGD) and sulfoquinovosyl diacylglycerol (SQD) to be modelled at the interface between PS II and the strongly-bound LHC II trimer (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These lipids presumably help to anchor the strongly-bound LHC II trimers to the PSII complex in plants. In the spinach structure (3JCU) there were no cofactors modelled at these positions; however, in the pea (5XNL) and spruce (8C29) structures, there were alternate lipids modelled at these positions. Fitting SQD and DGD into the pea or spruce maps or fitting of the alternate cofactors into the Arabidopsis map resulted in poor fits (Supplementary Fig.\u0026nbsp;4\u0026ndash;5). An α-tocopherol molecule is modelled in this region in the spruce structure (8C29). We also observe a density at a similar position which fits α-tocopherol; however, without experimental results to confirm the presence of an α-tocopherol molecule in Arabidopsis PS II, we chose not to include it in our model (Supplementary Fig.\u0026nbsp;6).\u003c/p\u003e \u003cp\u003eOur structure includes 1202 modelled water molecules, in comparison to 1076 waters in pea (5XNL), 280 in spruce (8C29) and none in spinach (3JCU). Excluding water molecules in the LHCII trimers, our structure resolves 1040 water molecules; approximately half of those identified in the current highest resolution structure of PS II from the cyanobacterium \u003cem\u003eT. vestitus\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e.\u003c/p\u003e\n\u003ch3\u003eWater networks at the PS II acceptor side\u003c/h3\u003e\n\u003cp\u003eAt the acceptor side of PSII, a water network has been identified in cyanobacterial structures that connects the stroma with the Q\u003csub\u003eB\u003c/sub\u003e site\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Our Arabidopsis structures finds several waters at similar positions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), as well as water positioned between Q\u003csub\u003eB\u003c/sub\u003e, D1:Tyr246 and D1:His215 (W63 in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). A water in this position has been hypothesized to participate in the hydrogen-bonding network which re-protonates the D1:His215 residue following donation of a proton to Q\u003csub\u003eB\u003c/sub\u003e\u003csup\u003e\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. A water in a similar position was also found in the recent high-resolution structure from \u003cem\u003eT. vestitus\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, suggesting that this re-protonation mechanism is conserved between cyanobacteria and plants. Alignment of our PS II structure to the structure from \u003cem\u003eT. vestitus\u003c/em\u003e (9EVX) showed that the D1 loop region, which forms part of the Q\u003csub\u003eB\u003c/sub\u003e binding pocket (D1:247\u0026ndash;267), is shifted in the Arabidopsis structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Additionally, the side chain of the D1:Ile248 residue is flipped relative to the \u003cem\u003eT. vestitus\u003c/em\u003e structure, creating space to accommodate two additional water molecules (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). This loop shift is consistent with molecular dynamics (MD) simulations\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, which indicate that this movement, (triggered by the release of Q\u003csub\u003eB\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e from the Q\u003csub\u003eB\u003c/sub\u003e pocket), can expand a water channel leading from the Q\u003csub\u003eB\u003c/sub\u003e pocket to the stroma (Supplementary Fig.\u0026nbsp;7A-B). B-factor analysis of the acceptor side region of PS II indicates that in our structure the B-factor of Q\u003csub\u003eB\u003c/sub\u003e is approximately 5 times higher than Q\u003csub\u003eA\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), while in the \u003cem\u003eT. vestitus\u003c/em\u003e structure the B-factor of Q\u003csub\u003eB\u003c/sub\u003e is only twice as high as Q\u003csub\u003eA\u003c/sub\u003e\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Further analysis confirms the reduced occupancy of Q\u003csub\u003eB\u003c/sub\u003e in our structure (Supplementary Fig.\u0026nbsp;7C). The observed structural differences between Arabidopsis and \u003cem\u003eT. vestitus\u003c/em\u003e are likely a result of the different light conditions during the sample preparation. Since the Arabidopsis PS II grids were prepared in dim light, a fraction of the Arabidopsis PS II particles would have an empty Q\u003csub\u003eB\u003c/sub\u003e binding pocket due to Q\u003csub\u003eB\u003c/sub\u003e reduction. Thus, the Arabidopsis Coulomb potential map around the Q\u003csub\u003eB\u003c/sub\u003e pocket likely represents an average position between the occupied Q\u003csub\u003eB\u003c/sub\u003e pocket and the empty Q\u003csub\u003eB\u003c/sub\u003e pocket conformations\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eWater networks around the MnCaO cluster\u003c/h3\u003e\n\u003cp\u003eExcluding the water ligands to the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster (W1-W4), fourteen water molecules were modelled in our structure within 9 \u0026Aring; of the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Nearly all these waters were clustered near the entrances of the O1, O4 and Cl1 channels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), which may suggest that having ordered water molecules in these positions is crucial for function. All the water molecules had an equivalent water in the \u003cem\u003eT. vestitus\u003c/em\u003e structure, except for the water modeled near the D1:Ala87 residue (W200), which is in the position of the polar headgroup of the D1:Asn87 side chain of cyanobacteria. This suggests that in plants, a water binds in place of the bulky asparagine side chain, fulfilling the role of the asparagine headgroup within the hydrogen-bonding network (Supplementary Fig.\u0026nbsp;8). Random noise begins to appear outside of the structure when the contour level of the locally sharpened map is reduced below 1.29 RMSD. At this contour level the map only shows density for two of the waters in the water wheel (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). However, a contour level of 0.49 RMSD there is a clear ring of density at the site of the water wheel (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). As the local resolution in the core is considerably higher than the global resolution, we felt confident in modelling these waters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D). This finding shows that the water wheel is conserved among species and supports the proposal that it plays a crucial role in water oxidation\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eWater/proton channels to the MnCaO cluster\u003c/h3\u003e\n\u003cp\u003eThe channels leading to the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster, and their water and hydrogen-bonding networks are pivotal for the stability and function of the oxygen evolving complex, as they regulate water access, enable controlled proton egress, and facilitate substrate water binding\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Comparing them in detail across species is crucial for understanding their specific functions and design principles. Here, we extend a previous study\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e by comparing the channels in our new Arabidopsis data with those in \u003cem\u003eT. vestitus\u003c/em\u003e and analyzing the positions of identified water molecules.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eO1 channel\u003c/h2\u003e \u003cp\u003eIt has been hypothesized that substrate water molecules reach the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster via the O1 channel\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. CAVER analysis showed that the O1 channel has three branches in the Arabidopsis structure, compared to two in \u003cem\u003eT. vestitus\u003c/em\u003e, and that these branches follow different routes to the surface of the protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). All branches of the O1 channel in Arabidopsis pass through a common bottleneck region near the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster. The only water molecule to be modelled in the O1 channels in the Arabidopsis structure was located in this bottleneck region (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). This indicates that this region is highly ordered, while the other water molecules in the O1 channel appear to be comparatively mobile, and thus more challenging to detect at our current resolution. The importance of this bottleneck is further highlighted by its conservation across species, as a corresponding water molecule was also found in the cryo-EM structures from \u003cem\u003eT. vestitus\u003c/em\u003e (9EVX) and in pea (5XNL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), as well as the x-ray crystallography structures from T.vestitus\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e (7rf1) and \u003cem\u003eThermosynechococcus vulcanus\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e (6dhe).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt has been proposed that this bottleneck region can act as a \u0026lsquo;water valve\u0026rsquo; that can open and close via allosteric actions at desired times during water oxidation, regulating water access to the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The conservation of the bottleneck region and the relatively immobile water within the bottleneck support this hypothesis. However, the model of PS II from \u003cem\u003eSynechocystis sp.\u003c/em\u003e PCC 6803 shows a different side chain orientation for D1:Glu329 and no water is modelled at the position shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCl1 channel\u003c/h3\u003e\n\u003cp\u003eThe Cl1 channel has been demonstrated to facilitate proton removal during water oxidation\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, facilitating proton transfer via the residues D1:Asp61, D1:Glu65 and D2:Glu312\u003csup\u003e9,13,23\u003c/sup\u003e. CAVER analysis showed that the Cl1 channel in Arabidopsis is highly similar to the Cl1 channel in \u003cem\u003eT. vestitus\u003c/em\u003e, conserving the positions of the critical proton-transfer residues and water molecules (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). This strong conservation indicates that the regulation of proton egress via the proton gate of the Cl1 channel is highly critical for water oxidation. Theoretical analyses and one experimental study indicate that the bottleneck residues and the D1-D61 side chain restrict water access to the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster through the Cl1 channel\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eO4 channel\u003c/h3\u003e\n\u003cp\u003eThe O4 channel has been hypothesized to be involved in the removal of protons from the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster during the S\u003csub\u003e0\u003c/sub\u003e\u0026loz;S\u003csub\u003e1\u003c/sub\u003e transition\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e However, it has also been suggested to supply water to the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe O4 channels in Arabidopsis and \u003cem\u003eT. vestisus\u003c/em\u003e initially follow a similar path; however, they diverge when the O4 channel passes through the extrinsic proteins, which differ between plants and cyanobacteria. Five water molecules were modeled within the O4 channel of Arabidopsis. These were not limited to one bottleneck region but were instead found at various places along the channel. This is consistent with previous observations made with cyanobacterial structures that the entire O4 channel is relatively narrow\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e.\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. In Arabidopsis, the bottleneck of the channel has a diameter of 1.6 \u0026Aring; compared to an average diameter of only 3 \u0026Aring;, indicating water mobility is restricted along the entire channel length in the O4 channel, as compared to a specific \u0026lsquo;water valve\u0026rsquo; in the O1 channel. This narrow width is expected to restrict water motion and, thus, the O4 channel appears better suited for proton transfer than water delivery.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe structure presented here represents a significant improvement over previously published structures of higher plant PSII, both in resolution and completeness, including our previous Arabidopsis PS II structure (7OUI)\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. By omitting digitonin and lowering the pH during PS II extraction, we were able to obtain intact PS II particles, leading to a complete structure of active, mature PS II. It is interesting to note that the PS II particles used for the spruce structure (8C29) were isolated at pH 7.5 and lacked PsbJ, PsbP, PsbQ and the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster, despite the absence of digitonin, suggesting that the choice of pH is critical for retaining the extrinsic subunits and the Mn\u003csub\u003e4\u003c/sub\u003eCaO\u003csub\u003e5\u003c/sub\u003e cluster.\u003c/p\u003e \u003cp\u003eIn all the higher plant structures, including the one presented here, the lipids connecting the CP47 subunit to the s-LHC II trimer had a different identity. As the identity of the lipids at this position may affect the strength of the LHC II trimer binding to the core and since LHC II trimers can dissociate from the core in high light conditions\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, we hypothesize that the identity of these lipids may modulate this protective mechanism.\u003c/p\u003e \u003cp\u003eSimulations of the acceptor side of PS II provide evidence that the structure of the Q\u003csub\u003eB\u003c/sub\u003e pocket changes when it is vacated\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Compared to the \u003cem\u003eT. vestitus\u003c/em\u003e structure (9EVX) our Arabidopsis structure had a shifted position for the D1:247–267 region, along with unusually high B-factors. This is likely the result of combining data of PSII particles that have Q\u003csub\u003eB\u003c/sub\u003e bound with a significant fraction of particles lacking Q\u003csub\u003eB\u003c/sub\u003e. This finding supports calculations showing movement of this D1 loop region following the dissociation of Q\u003csub\u003eB\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e from the Q\u003csub\u003eB\u003c/sub\u003e binding pocket\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur present data is most consistent with one substrate water delivery channel, the O1 channel, and two proton release channels, one facilitating proton release during the S\u003csub\u003e0\u003c/sub\u003e-S\u003csub\u003e1\u003c/sub\u003e transition (O4 channel) and the other during the S\u003csub\u003e2\u003c/sub\u003e-S\u003csub\u003e3\u003c/sub\u003e and S\u003csub\u003e3\u003c/sub\u003e-S\u003csub\u003e0\u003c/sub\u003e transitions (Cl1 channel). The O1 substrate water delivery channel has a short, highly conserved section up to the single point water valve that likely opens and closes water access at the right moments in the mechanism via allosteric control\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. After this bottleneck, the only requirement is that water can freely reach this point and thus it can vary dramatically between species without functional consequences. The design of the two proton channels is very different: the O4 channel has an extended line of well-ordered water molecules who’s H-bonding pattern has been proposed to open/close the proton egress pathway\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. By contrast, the Cl1 channel is less restricted, and the proton egress pathway involves several conserved charged amino acids and is regulated by proton gate residues that likely open by electrostatic control\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. We thus speculate that the Cl1 proton exit path can only operate in the higher S states when the positive extra charge is present as in the S\u003csub\u003e2\u003c/sub\u003e and S\u003csub\u003e3\u003c/sub\u003e states. In these states, the Cl1 channel is more efficient for proton release than the O4 channel, while in the neutral S\u003csub\u003e0\u003c/sub\u003e state, the O4 channel can be used for proton egress.\u003c/p\u003e \u003cp\u003eThe present advancement in the resolution of plant PS II has allowed a level of comparison to cyanobacterial PS II that was previously impossible. Through these comparisons, it is possible to determine which features of the protein and water network of PS II are essential, providing insight into design principles of photosynthesis and enzyme catalysis as a whole.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Methods","content":"\u003ch2\u003ePhotosystem II extraction, purification and activity assay\u003c/h2\u003e\u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Columbia-0) plants were grown for 8 weeks at constant temperature (20 \u003csup\u003eo\u003c/sup\u003eC) and humidity (70%), and day/night periods of 8 h light/16 h dark. The growth of these plants complied with the local and national regulations and the permission was given from Jordbruksverket-Växtkontrollenheten (Dnr 4.6.20–6365/15). The leaves of the plants were harvested after 16 hours of dark, and BBY membranes\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e were extracted from the leaves as described in Chen et al., 2019\u003csup\u003e47\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBBY membranes were washed with wash buffer (25 mM MES pH 6.3, 10 mM NaCl, 5 mM CaCl\u003csub\u003e2\u003c/sub\u003e) and resuspended in wash buffer at a concentration of 1 mg mL\u003csup\u003e− 1\u003c/sup\u003e Chlorophyll. BBY membranes were then diluted to 0.5 mg.mL\u003csup\u003e− 1\u003c/sup\u003e with 0.6% (w/v) n-dodecyl-β-maltoside (β-DM) in wash buffer, giving a final concentration of 0.3% β-DM and left with slow spinning for 1 min. Insoluble material was pelleted by centrifuging at 16,000 x g for 5 min, before transferring the supernatant and spinning again at 20,000 x g for 10 min. The supernatant was loaded onto the top of sucrose gradients which had been formed by the freeze-thawing method (25 mM MES pH 6.3, 0.5 M sucrose, 1 M betaine, 10 mM NaCl, 5 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 0.01% (w/v) β-DM (Anatrace, U.S.A). Sucrose gradients were centrifuged at 141,000 x g for 18 h, and the resulting bands were separated. Oxygen evolution measurements were made on the separated bands using a Clark-type electrode (Hansatech). The bands were measured at a chlorophyll concentration of 10 µg mL\u003csup\u003e− 1\u003c/sup\u003e of chlorophyll with 0.2 mM PPBQ (Phenyl-p-benzoquinone) and of 1 mM K\u003csub\u003e3\u003c/sub\u003eFeCN\u003csub\u003e6\u003c/sub\u003e added as electron acceptors using saturating light (Supplementary Fig.\u0026nbsp;9).\u003c/p\u003e\u003ch2\u003eSample preparation and electron microscopy\u003c/h2\u003e\u003cp\u003eC\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eM\u003csub\u003e2\u003c/sub\u003e and C\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e complexes were concentrated using 100 kDa molecular weight cut-off spin filters (Amicon) and then washed several times with pre-freezing buffer (50 mM MES pH 6.3, 20 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.6 M Betaine, 0.03% (w/v) β-DM) before finally concentrating to a final concentration of 1.6 mg Chl mL\u003csup\u003e− 1\u003c/sup\u003e. 4 µL of sample was loaded onto glow-discharged (30s at 50 mA) Quantifoil grids R 1.2/1.3 Cu 300 (Quantifoil Micro Tools) in dim room light. The grids were then plunge frozen in liquid ethane using an FEI Vitrobot MkIV (Thermo Fisher Scientific) at 100% humidity, 4 \u003csup\u003eo\u003c/sup\u003eC, and a blot force of -5, a wait time of 1 s and a blotting time of 5 s. Automated data collection was performed using EPU software on a Titan Krios G2 transmission electron microscope operating at 300 kEV (Thermo Fisher Scientific) at the Umeå Core Facility for Electron Microscopy, a node of the Swedish National Cryo-EM facility. The Titan Krios was equipped with Falcon 4 direct electron detectors and a Selectris energy filter. In total 16,450 movies were collected, which each contained 40 frames. The movies were collected at a pixel size of 0.704 Å, a total dose of 40 electrons/Å\u003csup\u003e2\u003c/sup\u003e and defocus values that ranged from − 0.8 to -2.0.\u003c/p\u003e\u003ch2\u003eData processing\u003c/h2\u003e\u003cp\u003eThe dataset was processed using cryoSPARC software. Motion and contrast transfer function corrections were performed before template free picking was used to identify particles. 3,054,062 particles were initially identified, before several rounds of 2D classification reduced the number of “good” particles to 322,522. \u003cem\u003eAb initio\u003c/em\u003e 3D reconstruction was used to generate 6 different 3D volumes, which were then refined using heterogeneous refinement. The particles used to generate the best-looking class went through additional filtering using 2D classification and multi-class \u003cem\u003eab initio\u003c/em\u003e 3D reconstruction until a final volume was generated using 72,301 particles and refined using homogeneous refinement and non-uniform refinement with imposed C2 symmetry. The final volume had an estimated global resolution of 2.44 Å, using the gold standard FSC (Supplementary Fig.\u0026nbsp;10). Finally, the map was sharpened, using Phenix to perform local sharpening with the Autosharpen function.\u003c/p\u003e\u003ch2\u003eModel building\u003c/h2\u003e\u003cp\u003eThe model for spinach photosystem II (3JCU) was used as a starting point and manually fitted into the map using UCSF ChimeraX software (version 1.7) \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. COOT (version 0.9.8.92)\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e was used to perform the relevant mutations to change the \u003cem\u003eSpinacia oleracea\u003c/em\u003e protein sequences to the corresponding \u003cem\u003eArabidopsis thaliana\u003c/em\u003e sequences. COOT was then used to refine the model to fit into the sharpened map. Several rounds of automatic fitting and manual fitting/checking were performed to optimize the fitting of the model into the map densities. Final refinement was performed using the Real-space refinement function in Phenix\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, with manual checking in COOT to confirm or correct the automatic refinement. All structures in the figures were prepared with ChimeraX or Pymol. Details of the data collection and model building are shown in Supplemental tables 1–3.\u003c/p\u003e\u003ch2\u003eWater channel calculations\u003c/h2\u003e\u003cp\u003eThe channels connecting the Manganese cluster to the exterior of the protein were calculated using CAVER software\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e (version 3.0.3), which is a plugin for PyMOL\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. For most of the channels, standard settings were used: probe radius 0.9, shell depth 4, shell radium 3, clustering threshold 3.5. For the Cl1 channel, it was necessary to reduce the probe radius to 0.6.\u003c/p\u003e\u003ch2\u003eB-factor analysis\u003c/h2\u003e\u003cp\u003eThe structure was refined using REFMAC5\u003csup\u003e53\u003c/sup\u003e, implemented in Servalcat, with the two unsharpened half-maps and the required mask file. Figures related to the analysis were prepared using PyMOL\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThe cryo-electron microscopy data was collected at Ume\u0026aring; Core Facility for Electron Microscopy, a node of the SciLifeLab National Cryo-EM Facility, funded by the Knut and Alice Wallenberg Foundation, the Erling-Persson Foundation,the Kempe Foundation, SciLifeLab, Stockholm University, and Ume\u0026aring; University. This work was supported by Vetenskapsr\u0026aring;det grant 2020-03809 (J.M.); Carl Tryggers Foundation (CTS 19.324) (W.P.S.); Kempe (JCK-2030 2021-2023) (W.P.S. and J.M.); German Research Foundation (DFG) grant through the Collaborative Research Center SFB1078 (Humboldt Universit\u0026auml;t zu Berlin, grant no.TP A5); SFB 1507 \u0026ldquo;Membrane-associated protein assemblies, machineries, and\u003cbr /\u003e supercomplexes\u0026rdquo; (R.H.) and the Lawski foundation (date 240612) (A.G. and J.F.). We would also like to acknowledge A. Bhomwick, V.K. Yachanda, J. Yano and J.F. Kern for their helpful discussions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRenger, G., Renger, T. (2008). Photosystem II: The machinery of photosynthetic water splitting. Photosynth. Res., 98(1\u0026ndash;3), 53\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShevela, D., Kern, J. F., Govindjee, G., Messinger, J. (2023). 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Crystallogr. 67, 235\u0026ndash;242.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5656066/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5656066/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhotosystem II (PSII) is a large membrane-bound protein complex that is essential for oxygenic photosynthesis in plants and cyanobacteria. While there are several high-resolution structures of PS II from cyanobacteria, very few PS II structures have been reported from higher plants. Here, we present the first high-resolution structure of an intact and active PS II from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e at a resolution of 2.44 \u0026Aring;, which to date represents the highest resolution structure of PS II from any land plant. The improved resolution allowed for the modeling of cofactors which have not been previously seen in higher plant PS II structures. Importantly, we were able to identify many water molecules within the PS II structure, including waters around the water-splitting manganese cluster, in the bottleneck regions of the water/proton channels, and near the non-heme iron on the acceptor side. Structural differences between cyanobacterial and plant PS II are discussed.\u003c/p\u003e","manuscriptTitle":"Structure of the intact C2S2-type Photosystem II-LHCII supercomplex from Arabidopsis thaliana at 2.44 Å","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-09 08:54:47","doi":"10.21203/rs.3.rs-5656066/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6f27bd0c-f02b-4ac5-92df-2c772751ddc8","owner":[],"postedDate":"January 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":42568402,"name":"Biological sciences/Plant sciences/Photosynthesis/Photosystem II"},{"id":42568403,"name":"Biological sciences/Biological techniques/Structure determination/Electron microscopy/Cryoelectron microscopy"}],"tags":[],"updatedAt":"2025-02-27T15:51:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-09 08:54:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5656066","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5656066","identity":"rs-5656066","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}