Algal photosystem I dimer and high-resolution model of PSI-plastocyanin complex

Photosystem I (PSI) enables photo-electron transfer and regulates photosynthesis in the bioenergetic membranes of cyanobacteria and chloroplasts. Being a multi-subunit complex, its macromolecular organization affects the dynamics of photosynthetic membranes. Here we reveal a chloroplast PSI from the green alga Chlamydomonas reinhardtii that is organized as a homodimer, comprising 40 protein subunits with 118 transmembrane helices that provide scaffold for 568 pigments. Cryogenic electron microscopy identified that the absence of PsaH and Lhca2 gives rise to a head-to-head relative orientation of the PSI–light-harvesting complex I monomers in a way that is essentially different from the oligomer formation in cyanobacteria. The light-harvesting protein Lhca9 is the key element for mediating this dimerization. The interface between the monomers is lacking PsaH and thus partially overlaps with the surface area that would bind one of the light-harvesting complex II complexes in state transitions. We also define the most accurate available PSI–light-harvesting complex I model at 2.3 Å resolution, including a flexibly bound electron donor plastocyanin, and assign correct identities and orientations to all the pigments, as well as 621 water molecules that affect energy transfer pathways.

Photosystem I (PSI) enables photo-electron transfer and regulates photosynthesis in the 22 bioenergetic membranes of cyanobacteria and chloroplasts. Being a multi-subunit complex, its 23 macromolecular organization affects the dynamics of photosynthetic membranes. Here, we reveal 24 a chloroplast PSI from the green alga Chlamydomonas reinhardtii that is organized as a 25 homodimer, comprising 40 protein subunits with 118 transmembrane helices that provide scaffold 26 for 568 pigments. Our cryo-EM structure identifies that the absence of PsaH and Lhca2 gives rise 27 to a head-to-head relative orientation of the PSI-LHCI monomers in a way that is essentially 28 different from the oligomer formation in cyanobacteria. The light-harvesting protein Lhca9 is the 29 key element for mediating this dimerization. The interface between the monomers is lacking PsaH, 30 and thus partially overlaps with the surface area that would bind one of the LHCII complexes in 31 state transitions. We also define the most accurate available PSI-LHCI model at 2.3 Å resolution, 32 including a flexibly bound electron donor plastocyanin, and assign correct identities and 33 orientations of all the pigments, as well as 621 water molecules that affect energy transfer 34 pathways. 35 36

Main 37
A chloroplast PSI of green algae consists of the core complex and three antenna modules: inner 38 belt, outer belt, and Lhca2:Lhca9 heterodimer, which together comprise 24 subunits 1-4 . As a short-39 term light acclimation mechanism in response to fluctuating illumination and anoxia, the algal PSI 40 additionally associates with two LHCII trimers 5,6 . Structural studies have shown that the 41 oligomeric state of a chloroplast PSI is a monomer, due to the presence of the subunit PsaH, 42 whereas in cyanobacteria structures of dimers 7-10 and trimers 11 were also reported. Cyanobacterial 43 PSI oligomerizes via direct contacts between subunits PsaI and PsaL, however such an association 44 has been ruled out for a chloroplast PSI due to structural constraints of PsaH presence that imposes 45 an apparent rigidity 12,13 . Yet, recent structural studies of PSI from a chloroplast of a salt-tolerant 46 alga suggested that its functional core may vary more than previously believed 14 . Particularly, 47 functional PsaH-free particles were found, thus showing a potential architectural plasticity of PSI 48 in response to the ecological environment. On the macromolecular level, an atomic force 49 microscopy analysis of a plant thylakoid membrane showed that when its architecture is altered 50 upon transition from darkness to light, larger inter-membrane contacts are formed, leading to a 51 reduced diffusion distance for the mobile electron carriers 15 . The membrane architecture in dark-52 and light-adapted membranes contains ordered rows of closely packed PSI dimers, which are more 53 abundant in the dark state 15 . Similarly, closely associated PSI-LHCI complexes were detected in 54 plants by negative stain electron microscopy 16 , and dimers were found in a subpopulation of PSI 55 from a temperature-sensitive PSII mutant alga 17 . This suggests that reversible PSI dimer formation 56 may have a physiological role in thylakoid membrane structure maintenance in chloroplasts. 57 However, very little is known about PSI-LHCI dimers and information on their structures is 58 lacking. In the absence of high resolution data, no evidence is available on composition, elements 59 regulating and mediating dimerization, and how the arrangement would differ from the 60 cyanobacterial counterparts. 61 62

Structure determination 63
We grew C. reinhardtii cells containing a His-tag at the N-terminus of PsaB in low light and under 64 anoxic conditions (see Methods). The thylakoid membranes were solubilised with n-dodecyl-α-D-65 maltoside (α-DDM), followed by affinity purification, crosslinking via the chemically activated 66 electron donor plastocyanin (Pc) and sucrose density gradient centrifugation (Extended Data Fig.  67 1). Two PSI fractions were detected on the sucrose gradient, and 2D polyacrylamide gel 68 electrophoresis (native/reducing 2D-PAGE) of isolated thylakoids indicated the presence of PSI 69 dimers (Extended Data Fig. 2 and Supplementary Table 1). The heavier green band on the gradient 70 was subjected to single-particle cryo-EM analysis (Supplementary Table 2). We used 2D 71 classification to separate PSI dimers from monomers in a reference free manner, followed by 3D 72 classification leading to a subset of 14,173 particles, which were refined to an overall resolution of 73 2.97 Å by applying C2 symmetry (Extended Data Fig. 3). PSI dimers were also found in 2D class 74 averages in a dataset recorded from a sample without the use of crosslinker. Upon symmetry 75 expansion, the resolution was further improved to 2.74 Å (Extended Data Arrangement of the pigments in the outline of the map: chlorophylls green (Mg yellow), luteins 101 blue, beta-carotenes red, violaxanthin purple, and neoxanthin pink c, Overall view along the 102 membrane. 103 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 25, 2022. ; https://doi.org/10.1101/2021.08.30.458224 doi: bioRxiv preprint

Structural basis for PSI dimerization 104
The structural basis for the algal chloroplast PSI dimerization is fundamentally different from 105 cyanobacteria (Fig. 2a). In cyanobacteria, PSI dimerises via the stromal region of PsaL 7-10 and 106 trimerises via the lumenal C-terminus of PsaL, assisted by PsaI 11 . In our structure of the chloroplast 107 PSI-LHCI dimer, neither PsaL nor PsaI interacts with each other between the neighbouring units. 108 Instead, PsaH that normally preserves a monomer is not present, and Lhca9 with its associated 109 cofactors acts as a symmetrical linker between the monomers, highlighting the importance of the 110 light-harvesting antenna proteins for regulation of the macro-organisation. Lhca9 is distinct among 111 the light-harvesting proteins in our structure due to a truncated loop between helices A and C, and 112 lack of the associated chlorophyll 6 . As a result, it contains the fewest chlorophylls among Lhcas 113 (Supplementary Table 3). Based on this difference, we rationalised how Lhca9 allows for 114 dimerization, as a longer AC-loop would clash with the neighbouring PsaB (Extended Data Fig.  115 5). 116 The two Lhca9 copies tether the PSI monomers in a head-to-head fashion, resulting in a 340-Å 117 long structure (Fig. 1, Fig. 2b

Implications of PSI dimerization 146
The specific interactions between the monomers are enabled due to unoccupied positions of PsaH 147 and Lhca2. Since PsaH is also required for the lateral binding of LHCII to the PSI core in state 148 transitions 5,6 , we next compared the structure of PSI-LHCI dimer to the state transition complex 149 (Fig. 3). The superposition shows that Lhca9 from the neighbouring monomer is positioned in the 150 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  membrane, where Lhca2 resides in PSI-LHCI-LHCII, and their three transmembrane helices would  151 overlap with each other (Fig. 3b). The presence of the PsaH transmembrane helix is not compatible  152 with the Lhca9 2 -associated cofactors CLA9, LMG852, BCR9 that extend from the neighbouring 153 monomer in the dimer. In addition, the superposition shows that there would be a clash between 154 PsaG and Lhca1 of the inner belt with one of the LHCII trimers, but not the other (Fig. 3b). Since 155 Lhca2 and PsaH are absent, the structure of the algal PSI dimer would not facilitate LHCII binding 156 at this position. However, our 2D-PAGE indicated a comigration of LHCII polypeptides with the 157 dimer fraction, and therefore a structural adaptation cannot be excluded (Extended Data Fig. 2). 158 The antagonistic relationship of Lhca9 2 and Lhca2, and the assembly state of PsaH might further 159 reflect a regulation of PSI dimerization (Fig. 3a). 160 PsaH is a 11-kDa transmembrane protein that is imported into chloroplasts and peripherally 161 associates To further extrapolate potential conformational changes during the dimerization of PSI, we applied 205 multi-body refinement analysis of the PSI dimer using the two monomers as bodies (Extended Data 206 Fig. 9). The analysis indicated no distinct conformational states, but instead revealed continuous 207 motions in the three eigenvectors describing a relative movement of the monomers in relation to 208 each other (Extended Data Fig 9a). The intrinsic flexibility is dominated by combinations of all 209 three rotations of one monomer with respect to the other up to 13° (Extended Data Fig. 9b-d). 210 Therefore, excitation energy transfer between the PSI monomers in the dimeric scaffold would also 211 depend on degrees of rotation around the identified pivot points. Specifically, three chlorophylls 212 are found within a potential cross-monomer excitation-sharing: CLA807 (PsaB), CLA604 213 (Lhca9 2 ), CHL606 (Lhca9 2 ), and the distance between them is ~20 Å in the consensus map ( Fig.  214 2). While such a positioning might suggest direct coupling, the multi-body analysis indicates 215 considerable variability (Extended Data Fig. 9e,f). Therefore, similar to the cyanobacterial PSI 216 dimer, an excitation coupling between the two monomers is less favourable in vitro, and this is 217 consistent with measurements of 77 K fluorescence spectra that showed only a minor shift between 218 monomer and dimer (Extended Data Fig. 1). However, in vivo the observed PSI-LHCI dimer 219 conformation, and therefore the distance between the chlorophylls at the interface, could also be 220 affected by a local membrane curvature. 221 222

High resolution features and solvation of PSI 223
In our PSI monomer reconstruction, the resolution in the core is ~2.1 Å, and in the LHCI inner belt 224 2.1-2.5 Å, revealing unprecedented structural details of chlorophylls, carotenoids, and 621 water 225 molecules ( to Chl a 617, respectively (Fig. 4b). The newly modeled beta-carotene 622 is in Lhca9 and could 234 be identified due to structural stabilization of the interface region in the dimer (Fig. 4b). Since chl 235 b limits free diffusion of excitation energy 42 , some of the new assignments affect the energy 236 pathways between the antenna proteins. Together with the new structural data, this allowed us to 237 produce a more accurate map of the energy channelling in PSI based on the new model (Fig. 4a). 238 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Another striking feature of the high resolution cryo-EM map is resolvable density for multiple 239 newly detected water molecules, which particularly aided in modeling the coordination of 240 chlorophylls (Fig. 4c, Supplementary movie 1). Thus, we report the most complete available 241 experimental picture of a chemical environment for chlorophyll binding (Supplementary Table 3). 242 Particularly, it allows distinguishing between mono-and di-hydrated forms, which largely escaped 243 detection by X-ray crystallography (Extended Data Fig. 12). This is mechanistically important 244 because the di-hydrated derivative is chemically more stable, as illustrated by quantum chemical 245 calculations 43 . We observe that, other than the previously reported CLA824 20 , only two waters 246 can be involved in penta-coordinated Mg for all the chlorophylls. Remarkably, waters play a 247 coordinative role for most of Chl b, for which the relative ratio of water coordination is four times 248 higher than for Chl a (Supplementary Table 3). The difference between Chl a and Chl b is a methyl 249 versus a formyl group, thus water serves as a hydrogen bond donor to the latter, while it also 250 interacts with charged/polar protein residues or lipids. Therefore, the immediate surrounding of 251 Chl b molecules is more enriched with non-protein material than previously thought, which plays 252 a role in tuning the photophysics and the transport properties of excitation energy in PSI. Together, 253 the presented model now allows for comparison of PSI phylogenetic conservation also on the level 254 of chlorophyll coordination and solvent positioning. 255 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  phylloquinones purple, iron-sulphur clusters yellow-red. 263 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 25, 2022. ; https://doi.org/10.1101/2021.08.30.458224 doi: bioRxiv preprint

Structure of PSI:Pc complex 264
On the lumenal side of PSI, we observed a density corresponding to the associated electron donor 265 Pc, which binding has been stabilised by crosslinking (see Methods). The bound Pc is found on 266 PSI monomers and dimers. Signal subtraction, followed by focused 3D classification, allowed us 267 to rigid body fit a model for Pc into the density at the local resolution of ~3.5 Å (Extended Data 268 Fig. 3). We then performed flexible fitting using self-similarity-restraints in Coot 44,45 . With respect 269 to the PSI-Pc interactions, comparison between our model with a plant counterpart 46,47 revealed 270 two main differences (Fig. 5). In C. reinhardtii, the negatively charged residues of the Pc acidic 271 patch are shifted by ~5 Å due to the missing residues P58-E59, and therefore, the interaction with 272 K101 of PsaF is weakened (Fig. 5a,b) Thylakoids were collected from the step gradient interphases with a Pasteur pipet, diluted four 324 times with 5 mM HEPES-KOH pH = 7.5 and centrifuged at 21,5000 rpm for 20 min (Beckman 325 Coulter JA 25.50). 326 Isolated thylakoids were set to 1 mg chl mL -1 in 5 mM HEPES-KOH pH = 7.5 and solubilized by 327 addition of an equal volume of 2 % α-DDM for 10 min. Unsolubilized material was separated by 328 centrifugation. The supernatant was diluted four times to 125 µg chl mL -1 and 0.25 % α-DDM. The 329 sample was loaded onto a TALON metal affinity column (1 mL resin mg chl -1 ) in 5 mM HEPES-330 KOH pH = 7.5, 100 mM NaCl, 5 mM MgSO4, 10 % glycerol at a flow rate of ~0.5 mL min -1 . The 331 column was washed with 10 times the bed volume of 5 mM HEPES-KOH pH = 7.5, 100 mM NaCl, 332 5 mM MgSO4, 10 % glycerol, 0.02 % α-DDM at a flow rate of ~1 mL min -1 . A second wash was 333 performed with 10 times the bed volume of 5 mM HEPES-KOH pH = 7.5, 100 mM NaCl, 5 mM 334 MgSO4, 10 % glycerol, 0.02 % α-DDM and 5 mM imidazole at a flow rate of ~1 mL min -1 . The 335 PSI was eluted with 5 mM HEPES-KOH pH = 7.5, 100 mM NaCl, 5 mM MgSO4, 10 % glycerol, 336 0.02 % α-DDM and 150 mM imidazole. The PSI was concentrated with a spin column (regenerated 337 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made (C18 PepMap 100, 300 µM x 5 mm, 5 µm particle size, 100 Å pore size; Thermo Fisher Scientific) 374 and desalted for 3 min at a flow rate of 15 µL min -1 using eluent A1. Subsequently, the trap column 375 was switched in-line with an Acclaim PepMap100 reversed phase column (75 µm x 50 cm, 2 µm 376 particle sizes, 100 Å pore size; Thermo Fisher Scientific) for peptide separation. The mobile phases 377 were composed of 0.1 % (v/v) formic acid in ultrapure water (eluent A2) and 80 % (v/v) 378 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made (un-binned) and processed in RELION using a box size of 700 pixel and 500 pixel for the dimer 446 and monomer, respectively. 3D Refinement followed by 3D classification was performed imposing 447 C2 symmetry for the dimer and C1 for the monomer. A subset of high quality particles was selected 448 for the dimer and monomer and subjected to 3D refinement which resulted in a resolution of 3.3 Å 449 for the dimer and 2.9 Å for the monomer. CTF refinement 67,68 followed by 3D refinement and 450 Bayesian polishing followed by another round of CTF refinement was performed for the dimer as 451 well as for the monomer. A final 3D refinement resulted in an overall resolution of 2.97 Å for the 452 dimer and 2.31 Å for the monomer. The resolution of the dimer could be further improved to 2.74 453 Å by using signal subtraction of one monomer followed by symmetry expansion and 3D refinement 454 applying C1 symmetry. 455 In order to increase the number of particles for classification on the Pc region, Dataset 2 was 456 collected from the same dimer band, but with a pixel size of 0.51 Å. The dataset was processed 457 with cryoSPARC 3.1.0 61 . After template picking 864,399 particles were extracted. With a small 458 subset of the extracted particles an Ab initio reconstruction was generated followed by 459 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Eigenvectors describing the motion. Ten maps for each of the three Eigenvectors which describe 473 about 78% of the motion in the data were printed out and the maps with the extreme positions 474 (map1 and 10) were used to fit the models that are shown in Extended Data Figure 8. A Python 475 script was used to estimate the distances between the chlorophylls at the dimer interface for each 476 particle in the data and to plot the results as histograms as depicted in Extended Data Figure 9e, To further analyse the identity of the corresponding pigment possible candidates were fitted and 492 compared. A carotenoid that fitted best in terms of density and chemical environment was then 493 selected. In case of luteins, the oxygen of the cyclohexane-ring was the main criterion for pigment 494 identity because it is involved in hydrogen bonding. For the Chl b identification the densities for 495 the aldehyde group needed to be present as well as the hydrogen bonding occurring with a water 496 molecule that are usually stabilized by other chlorophylls, lipids, and protein side chains. Water 497 molecules involved in pigment interactions were placed manually. All other water molecules were 498 picked by Coot 44,45 with the auto picking function followed by manual inspection and correction. 499 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 25, 2022. ; https://doi.org/10.1101/2021.08.30.458224 doi: bioRxiv preprint All high resolution features were modelled using Coot 44,45 until the model was completed. For all 500 modeling steps restraint files for pigments and ligands were used which were generated using the 501 Grade server (http://grade.globalphasing.org). Restraint files were adopted manually if it was 502 required. 503 For plastocyanin, a model was generated using SWISS model 71 . The model was then rigid body 504 fitted using Chimera. Rotamers were corrected for the residues that were allowed due to the better 505 local densities. Self-restraints in Coot were activated followed by flexible fitting into the density. 506 All models were refined using Real-Space-Refine from the PHENIX suite 72 using the Grade server 507 restraint files for the ligands and a distance .edit file which was generated by Ready-set in PHENIX. 508 Further, hydrogen atoms were added for refinement to the model using Ready-set. The refinement 509 protocol was optimized using different weight parameters. The refinement statistics are shown in 510 Supplementary Table 2. Multiple rounds of validation and model building were carried out using 511 MolProbity 73 and Coot 44,45 . For further validation the PDB Validation server was used 512 (https://validate-rcsb-2.wwpdb.org). 513 The structure was analysed using Coot and Chimera. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Notably, the presence of PsaH in the dimer sample, but its absence in the cryo-EM structure, 765 indicates that PsaH is only loosely attached to the PSI dimer and readily lost during the cryo-EM 766 analyses. 767 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made control conditions. b, low light and anoxic conditions. In the first dimension, multi-protein 770 complexes were separated in their native form by blue native PAGE. In the second dimension, 771 subunits of these protein complexes were separated by SDS-PAGE. "PSII complexes" refers to 772 PSII complexes of different molecular weight due to varying extents of LHCII association. 773 Labelled silver-stained spots were subjected to LC-MS/MS analysis. For convenience, the figure  774 includes a condensed list of representative subunits. For a list of all proteins identified in the 775 respective spots refer to Extended Data Detection of these small PSI and Lhca subunits via mass spectrometry depends on the presence of 824 only a few proteotypic tryptic peptides, which could be missed, as measurements were done in a 825 data dependent fashion (12 MS 2 per MS 1 ). 826 827 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made biochemically and structurally defined PSI states and assembling protein subunits. Subunit 830 composition for each state is outlined with the shape of a following state and corresponding color. 831 The divaricating of the assembly pathway takes place at the last stage (red background). The path 832 towards PSI monomer or dimer is dependent on presence/absence of PsaH and Lhca2. 833 834 835 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  vectors. e, f, The distance between the chlorophylls is plotted based on the relative motion from 840 the multi-body analysis. The y-axis shows the counts of particles that exhibit a certain distance. 841 842 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  with the corresponding density. Local resolution and map levels are indicated. 867 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

875
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 25, 2022. ; https://doi.org/10.1101/2021.08.30.458224 doi: bioRxiv preprint