Architecture of symbiotic dinoflagellate photosystem I–light-harvesting supercomplex in Symbiodinium

Domain: earth_science
Published: 2024-03-16
Editorial outcome: accept

Abstract
Symbiodinium are the photosynthetic endosymbionts for corals and play a vital role in supplying their coral hosts with photosynthetic products, forming the nutritional foundation for high-yield coral reef ecosystems. Here, we determine the cryo-electron microscopy structure of Symbiodinium photosystem I (PSI) supercomplex with a PSI core composed of 13 subunits including 2 previously unidentified subunits, PsaT and PsaU, as well as 13 peridinin-Chl a/c-binding light-harvesting antenna proteins (AcpPCIs). The PSI–AcpPCI supercomplex exhibits distinctive structural features compared to their red lineage counterparts, including extended termini of PsaD/E/I/J/L/M/R and AcpPCI-1/3/5/7/8/11 subunits, conformational changes in the surface loops of PsaA and PsaB subunits, facilitating the association between the PSI core and peripheral antennae. Structural analysis and computational calculation of excitation energy transfer rates unravel specific pigment networks in Symbiodinium PSI–AcpPCI for efficient excitation energy transfer. Overall, this study provides a structural basis for deciphering the mechanisms governing light harvesting and energy transfer in Symbiodinium PSI–AcpPCI supercomplexes adapted to their symbiotic ecosystem, as well as insights into the evolutionary diversity of PSI–LHCI among various photosynthetic organisms.

Introduction
Photosystem I (PSI) is a multi-subunit pigment-protein supercomplex that plays a crucial role in the light-driven photosynthetic electron transport of oxygenic photosynthesis. It accepts electrons from the Photosystem II (PSII)-mediated water oxidation delivered by plastocyanin or cytochrome c and transfers electrons to ferredoxin1. In eukaryotes, the PSI supercomplex consists of a core complex and peripheral light-harvesting complexes (LHCs). The LHCs associated with the PSI core (LHCI) capture and transfer light energy to the core complex, which is composed of the reaction center P700 and core antennae1. The structures of core subunits are highly conserved across cyanobacteria, algae, and plants2–15. In contrast, the LHCIs show high variability in their number, protein composition, and association of pigments among different oxygenic photoautotrophs, representing the intrinsic evolutionary and regulatory solutions utilized by photoautotrophic organisms to thrive in varying environmental niches16. Dinoflagellates are an ecologically and economically important group of unicellular eukaryotes, serving as primary producers widespread in various aquatic environments17–19. The plastids of the vast majority of photosynthetic dinoflagellates originated from a primordial endosymbiont20,21. One typical paradigm of dinoflagellate symbiosis is found in marine dinoflagellates belonging to the genus Symbiodinium. These photosynthetic endosymbionts form a crucial relationship with reef-building corals (zooxanthellae) in subtropical and tropical shallows, creating the trophic foundation for one of the world’s most diverse and productive marine ecosystems, namely coral reefs22–24. Such symbiotic systems exhibit remarkable productivity despite in nutrient-poor waters in which they thrive, attributed to the intimate mutualism between corals and photosynthetic Symbiodinium, which support the living of one-quarter to one-third of all marine species25.

The symbiotic relationship between Symbiodinium and corals is complex and delicate, and their mutualism is susceptible to environmental factors including temperature, light, and salinity26. The environmental stresses, such as high temperature and high light in the shallow ocean, could result in photoinhibition, pigment degradation, and oxidative damage in Symbiodinium, hampering nutrient flow between Symbiodinium and corals and consequently, leading to the devastating death of coral colonies27,28. Previous studies have suggested the role of Symbiodinium PSI–LHCI in photoprotection29,30. However, how Symbiodinium evolves the PSI–LHCI supercomplex to fulfill efficient energy and electron transfer, charge separation, and photoprotection remains poorly understood, and the lack of high-resolution structure has precluded a better understanding of Symbiodinium PSI–LHCI structure and function to ensure cell fitness in specific ecological environments.

Here, we solved the structure of PSI–LHCI from Symbiodinium using single-particle cryo-electron microscopy (cryo-EM) at a resolution of 2.8Å. Our study reveals distinct architectural features and excitation energy transfer pathways of Symbiodinium PSI–LHCI, providing insights into the mechanisms of light energy harvesting and transfer of the PSI–LHCI. These features may be essential for Symbiodinium cell fitness in specific symbiotic environments they form with other organisms.

Results
Overall structure
The PSI–LHCI supercomplex was purified from Symbiodinium sp. (Clade A) (Leading Tec, Shanghai, China) grown in a free-living state and the heavier green band was collected and analyzed (Supplementary Fig. 1, Supplementary Data 1). Using single-particle cryo-EM, we solved the PSI–LHCI supercomplex structure at an overall resolution of 2.8Å (Supplementary Fig. 2, Supplementary Table 1). Symbiodinium PSI–LHCI supercomplex comprises a PSI core complex and a peripheral antenna system containing 13 peridinin-Chl a/c-binding light-harvesting proteins (AcpPCs)31 that form a ring around the core (Fig. 1). Hereafter, the AcpPCs that are associated with PSI were named as AcpPCI. The PSI core is composed of 13 subunits, including PsaA–F, PsaI, PsaJ, PsaL, PsaM, and PsaR, as well as 2 previously unidentified subunits, PsaT (NCBI GenBank: PP196340) at the stromal surface and PsaU (NCBI GenBank: PP196339) at the lumenal surface (Fig. 1a–c, Supplementary Fig. 3a). To identify the amino acid sequences of PsaT and PsaU, small regions of the density maps of PsaT and PsaU with higher resolution were selected (Supplementary Fig. 3b, e), and amino acid sequences were manually speculated based on the selected map (Speculation sequence) (Supplementary Fig. 3c, f). Then, blastp against the transcriptome sequences was performed using the speculation sequences to identify potential sequences (Supplementary Fig. 3c, f). Each of the potential sequences was manually fitted into the selected map, and amino acids with distinguishable side chains (such as phenylalanine, tyrosine, tryptophan, arginine, and glycine) were used to identify the proper sequence from the candidate sequences (Supplementary Fig. 3b, e). The sequences were further confirmed by the suitability of the side chains for the density map in other regions (Supplementary Fig. 3d, g). Moreover, we assigned 196 Chl a, 20 Chl c, 41 diadinoxanthin (Ddx), 13 β-carotene, 16 dinoxanthin (Dx), 10 peridinin (Per), 2 phylloquinones, 3 Fe4S4 clusters, and 21 lipid molecules in the PSI–AcpPCI structure (Supplementary Table 2). Intriguingly, distinct from other reported PSI–LHCI structures, most of the PSI core subunits and half of the AcpPCI subunits possess extended terminal structures that span across both stromal and lumenal surfaces of the supercomplex (Fig. 1c, Supplementary Figs. 3–5). This specific organization, together with the membrane-protruded PsaT and PsaU subunits, enhances the longitudinal volume and inter-subunit interactions of the supercomplex, potentially promoting the structural integrity and functionality of Symbiodinium PSI–AcpPCI in symbiotic environment.

Structural features of the Symbiodinium PSI core
The Symbiodinium PSI core exhibits several specific structural properties compared to other reported PSI core complexes. Like diatom PSI, the Symbiodinium PSI lacks PsaK and PsaO but possesses the PsaR subunit, indicating its close evolutionary relationship with diatom PSI (Supplementary Fig. 6, Supplementary Table 3)3,4,13,14. In contrast to the relatively conserved transmembrane (TM) domains, notable variations are observed in the extrinsic loop structures of PsaA and PsaB and the terminal domains of the PsaD/E/I/J/L/M/R subunits (Supplementary Figs. 4, 5, 7).

The extended termini of PsaI/J/L/M/R, along with the PsaT subunit, form a protective layer that encompasses the stromal surface of the PSI core (Supplementary Fig. 8), resulting in reduced interactions between the reaction center and molecules in the stroma. This may mitigate the damage of reactive oxygen species to the reaction center and protect the pigments in the reaction center under high light. Moreover, these extended termini effectively create a specific PsaC/D/E-ferredoxin domain, presumably facilitating the binding of ferredoxin to PSI (Supplementary Fig. 8a–d). On the lumenal surface, the PsaU subunit spans across the edges of PsaA and PsaB without obstructing the binding sites for cytochrome c or plastocyanin. Such distinct structural features may enable efficient interactions and inter-molecular electron transfer between PSI and electron carriers (Supplementary Fig. 8e). Intriguingly, these long terminal extensions exhibit homologous sequences exclusive to dinoflagellate species, specifically in Symbiodinium (Supplementary Fig. 9). Likewise, the homologous sequences of PsaT and PsaU are exclusive in Symbiodiniaceae, with a high degree of conservation. These distinguishing characteristics provide evidence for a more diversified structure of the PSI core than was previously recognized.

The extended N-terminal domains of PsaL and PsaJ are situated on the stromal surface of PsaA, in proximity to the interface of PsaA and AcpPCIs (Fig. 2a). Intriguingly, both N-termini interact with each other, forming a molecular “thread” that stitches the stromal loops of AcpPCI-3, AcpPCI-5, and AcpPCI-6 together to strengthen the association of AcpPCI antennae (Fig. 2a, Supplementary Fig. 10). Moreover, their binding also lead to the conformational changes of PsaA at the N-terminal loop as well as the I2-V6, Q44-S47, Q164-S171, C276-D294, and Q362-I368 loops.

Interactions among the extended C-termini of PsaD and PsaI and the N-terminus of PsaR lead to formation of a “cap-like” structure on top of the stromal surface of PsaB (Supplementary Fig. 8). The C-terminus of PsaD directly binds to PsaB, extending across its surface and inducing the conformational changes of the PsaB V358-S366 and I148-S180 loops (Fig. 2b, Supplementary Fig. 11a). The PsaD C-terminus further extends to the outer edge of PsaB, forming interactions with AcpPCI-10 and AcpPCI-11, which may promote the physical association and energy transfer between AcpPCIs and the PSI core (Supplementary Fig. 11b). PsaD also has a longer N-terminal loop than those of reported PSI structures, forming interactions with the L628-V633 loop of PsaB (Supplementary Figs. 9, 11c). The extended termini of PsaI and PsaR interact with each other and form intense hydrogen bonds with adjacent subunits, thereby stabilizing their conformations (Fig. 2c, e; Supplementary Fig. 11d–f). The PsaI C-terminal extension may also adapt to a shortened N-terminus of PsaB (Fig. 2c, Supplementary Figs. 7, 11d).

The extended C-terminus of PsaM is positioned at the interface of the PSI core and AcpPCIs, and interacts with the I148-S180 and Y261-T270 loops of PsaB, the additional loop of PsaD, as well as adjacent AcpPCI-10 and AcpPCI-11 (Fig. 2d, Supplementary Figs. 5, 12a–c). The extended N-terminus of PsaM interacts with the N-terminus of PsaI and binds to the lumenal surface of PsaB, corresponding to the shortened PsaB C-terminus and T89-A102 loop (Fig. 2d, Supplementary Fig. 12d). The extended termini of PsaE and PsaF form intense interactions with adjacent subunits (Supplementary Fig. 12e, f). The lumenal loops of PsaB (A215-S223 and G429-S449) are shortened, mirroring their counterparts in PsaA (G213-L244 and A421-I431). This prevents interference with the extended N-terminus of PsaF and the lumenal loops of PsaR and AcpPCI-10 (Supplementary Fig. 12g).

The PsaT subunit (NCBI GenBank: PP196340) interacts with multiple core subunits, including PsaB−D, PsaL, and PsaI (Supplementary Fig. 13a, b). These associations may facilitate the stabilization of these extrinsic subunits. Interestingly, the PsaS subunit found in diatom PSI cores occupies a similar position to PsaT3,4; however, their structures exhibit low similarity (Fig. 2f). The association of the PsaU subunit (NCBI GenBank: PP196339) results in alternation of the G213-L244, A421-I431, and Y539-C561 loop structures of PsaA (Fig. 2g). Its N-terminus interacts with the extended N-terminus of PsaM, whereas its middle loop and C-terminus interact with AcpPCI-3 and AcpPCI-5/6, respectively, thereby mediating the association of AcpPCIs to the PSI core (Supplementary Fig. 13c).

Structures of AcpPCI subunits
Despite some similarities in subunit structure and association with PSI with the previously reported LHCs2–14, Symbiodinium AcpPCIs exhibit a notable level of distinctiveness. The binding positions of AcpPCI-6/7 are highly conserved across the LHCs of Symbiodinium, red algae, cryptophytes, and diatoms, whereas conformational shifts and rotations occur in the remaining AcpPCIs (Supplementary Fig. 14), owing to the specific core structures and loops of AcpPCIs. For instance, AcpPCI-1 rotates 180 degrees compared to its counterparts in red lineage PSI–LHCIs (Supplementary Fig. 14d, f). Since PsaK and PsaO are absent in the Symbiodinium PSI core, AcpPCI-3 and AcpPCI-4 occupy their positions and facilitate the binding of peripheral AcpPCIs, resembling diatom FCPI-3 and FCPI-43,4 (Supplementary Fig. 14c, f). However, AcpPCI-3 exhibits a slight shift compared to diatom FCPI-3, while AcpPCI-4 rotates about 90 degrees compared to FCPI-4, resulting in significant changes in the positions and orientations of AcpPCI-2/12/13 (Supplementary Fig. 14e, f). AcpPCI-5/8/9/10/11 shift toward to the PSI core. The specific conformation and arrangement enable the AcpPCIs to tightly associate with the PSI core, facilitating excitation energy flux from AcpPCIs to the PSI core.

The three TM helices (αA, αB, αC) of individual AcpPCIs are relatively conserved. In contrast, their loop structures exhibit flexibility (Fig. 3a–c; Supplementary Figs. 15, 16). AcpPCI-1/3/5/7/8/11 possess additional terminal loops that can interact with the PSI core. Similar extended terminal loops were also found in diatom FCPI-4/21/23/24 and green algal Lhca5/63,4,6,8,9, which primarily interact with adjacent LHCs. Phylogenetic analysis indicates that AcpPCIs have a close evolutionary relationship with diatom FCPIs, belonging to the Lhcr (AcpPCI-4/5/6/7/8/10/11) and Lhcf (AcpPCI-1/2/3/9/12/13) families (Supplementary Fig. 17). Lhcr-type AcpPCI-6/7/11 exhibit similar structures and share similarities with their counterparts in red lineage PSI–LHCIs, except for AcpPCI-7 that has an extended C-terminus and AcpPCI-11 with an extended N-terminus (Supplementary Fig. 18a). These extended regions interact with PsaA/J and the extended terminus of PsaD, respectively (Supplementary Figs. 11b, 19a, b). Lhcr-type AcpPCI-4/5/10 have short αB helices and loop structures that differ from AcpPCI-6/7/11 (Supplementary Fig. 18b). Notably, AcpPCI-5 possesses a longer N-terminus and AcpPCI-10 has a unique AB-loop compared to its counterparts in red lineage PSI–LHCIs, which associate with the extended termini of PsaL/J and the specific loop (A215-S223) of PsaB, respectively (Supplementary Figs. 12g, 18b, 19c, d). Lhcr-type AcpPCI-8 exhibits a high degree of similarity to its diatom counterpart, FCPI-8, except for an extended N-terminal loop that interacts with PsaA/B/E/F and the extended N-terminus of PsaR (Supplementary Figs. 18c, 19e). These interactions contribute to the proper association of AcpPCIs within the PSI–AcpPCI supercomplex.

The Lhcf-type AcpPCI-3 exhibits a similar structure to diatom FCPI-3 but possesses a very long C-terminal loop that is absent in FCPI-33,4 (Supplementary Fig. 18d). This extended loop spans along the interface between PsaA and AcpPCI-4/5, filling the gap resulting from the shortening of the loop structures of PsaA (G213-L244 and A421-I431) and enhancing the binding of AcpPCIs (Supplementary Fig. 19f). Lhcf-type AcpPCI-1/2/9/12/13 possess similar structures, including shorter αB helices compared to the Lhcr-type AcpPCIs (Supplementary Fig. 18e). AcpPCI-1, which is rotated by 180 degrees, possesses a longer C-terminal loop compared to its counterparts in red lineage PSI-LHCIs4,13,14. This longer C-terminal loop extends towards the lumenal side of the PsaI-PsaL region and occupies the position of the specific AB loops found in its counterparts (Supplementary Fig. 14d), providing an anchoring point for AcpPCI-1 (Supplementary Fig. 19g). AcpPCI-9 possesses a shorter αB helix and longer AB and BC loops compared to its Lhcr-type counterparts in red lineage PSI-LHCIs (Supplementary Fig. 18e). These structural differences likely contribute to the conformational shift of AcpPCI-9 towards the PSI core (Supplementary Fig. 14). Similarly, AcpPCI-13 also exhibits a shorter αB helix compared to diatom FCPI-13 (Supplementary Fig. 18e). The rotations of AcpPCI-12 and AcpPCI-13 relative to FCPI-12 and FCPI-13 result in their short αB helices facing AcpPCI-3, enabling them to occupy less space and position closer to AcpPCI-3 (Supplementary Fig. 14e).

Overall, Symbiodinium PSI–AcpPCI shares common architectural features with red algal PSI–LHCR, cryptophyte PSI–ACPI, and diatom PSI–FCPI; on the other hand, it also possesses specific characteristics in the protein structure and organization (Supplementary Fig. 14f). This provides insights into the structural diversity of PSI–LHCI in the red lineage. The unusually extended termini of AcpPCIs and core subunits ensure tight inter-molecular interactions of the PSI–AcpPCI supercomplex and result in the structural variations in the surface loops of PsaA and PsaB, providing the structural basis for the structural and functional integration and diversity of the PSI–AcpPCI supercomplex.

Pigment arrangement in PSI−AcpPCI
Notable differences are also found in the pigment arrangement Symbiodinium PSI–AcpPCI and reported red lineage PSI–LHCIs4,13,14 (Supplementary Fig. 20a, b). PSI–AcpPCI lacks 10 Chls in PsaA (A1-A10), 10 Chls in PsaB (B2, B4-B7, B10-B14), 2 Cars in PsaA (ACar2, ACar4), and 3 Cars in PsaB (BCar1-3) (Fig. 4a, b). Instead, 8 previously unidentified Chls (Chl832-833 in PsaA, Chl818 in PsaB and Chl403-404, Chl406-408 in PsaL) and a previously unidentified Car in PsaI appears. The absence of A1-A5 and ACar1-ACar2 in PasA is attributed to the conformational changes in the C276-D294 loop of PsaA at the stromal surface (Fig. 4c, Supplementary Fig. 20c). However, 3 Chls (Chl832 in PsaA and Chl407-408 in PsaL) are observed in the nearby positions close to the altered loop of PsaA and extended loop of PsaL. Similarly, A6-A8 are absent due to the conformational changes in the loops (A421-I431 and G213-L244) of PsaA at the lumenal surface (Fig. 4c, Supplementary Fig. 20d). A Chl (Chl833), close to A8, binds to the altered loop of PsaA. Furthermore, Symbiodinium PsaA loses two Chls because of the changes in the ligands of A9 and A10 from H205 and H315 in diatom PsaA to S182 and L272, respectively (Fig. 4d, Supplementary Fig. 20e).

The conformational changes in the loops of PsaB (Y261-T270 and S279-F284 at the stromal side, A215-S223 and G429-S449 at the lumenal side) also contribute to the dissociation of Chls and Cars (Fig. 4e, f). The shortening of the PsaB loop (S279-F284) and the presence of an extended N-terminal loop in AcpPCI-8 hinder the binding of B4 and B5 (Fig. 4e, Supplementary Fig. 20f). Changes in the loop region (G429-S449) result in the absence of B6 and B7 (Fig. 4e, Supplementary Fig. 20g). The absence of B4-B7, coupled with alternations in the surrounding residues, reduce hydrophobic interactions, resulting in the dissociation of B2, B11, BCar1 and BCar2 (Supplementary Fig. 20h, i). These changes also contribute to a decrease in the hydrophobic environment. The increased volume of the altered side chain and changes in their orientations further impede the binding of these pigments. Symbiodinium-specific substitutions in contrast to their diatom counterpart, which weaken the hydrophobic and hydrogen-bond interactions, along with the increased volume of the altered side chains, hinder the binding of B10 and B12-B14 (Fig. 4f, Supplementary Fig. 20j). Additionally, the substitution of V184 with F196 in Symbiodinium, in conjunction with the absence of B10, induces a confirmational flipping of the head of Car838, potentially facilitating the binding of the Chl818 (Fig. 4f). The loss of BCar3 is caused by the shortening of the lumenal loop (A215-S223) of PsaB, which disturbs the hydrophobic environment (Supplementary Fig. 20k). Furthermore, changes in the surrounding residues result in the conformational shifts of Chl810, Chl817, and Chl829 (Supplementary Fig. 20l, m).

Twenty Chl-binding sites and thirteen Car-binding sites were identified in AcpPCIs, out of which twelve Chl-binding sites (301-312) and five Car-binding sites (401-405) are conserved in the reported red lineage LHCIs2–4,13,14 (Fig. 3d, e, Supplementary Table 4). Other than the conserved sites, eight additional Chl sites (313-320) and eight additional Car sites are distributed across different AcpPCIs (Supplementary Fig. 21a–f). These specific Chl/Car-binding sites are located in the interface region between AcpPCIs and between AcpPCIs and the PSI core subunits (Supplementary Fig. 21e–f), suggesting their important role in mediating inter-subunit energy transfer. Interestingly, some Chl-binding sites, such as 301, 303, 304, 306 and 310, can accommodate either Chl a or Chl c (Supplementary Fig. 21b, c). Most AcpPCIs contain one or two Chl c, except for AcpPCI-9, which possesses five Chl c molecules (Supplementary Fig. 21e). The high content of Chl c in AcpPCI-9 may facilitate energy transfer from AcpPCI-9 to adjacent subunits, as Chl c has higher energy than Chl a3,7,8,32. All AcpPCIs feature a potential red-shifted Chl 305/306 pair located at the same positions as other LHCIs2–14,32. The PSI–AcpPCI complex contains Ddx and Dtx, as confirmed by HPLC analysis (Supplementary Fig. 1e). Since Ddx and Dtx share similar structures, the putative binding sites for Dtx found at a low level were assigned as for Ddx in the PSI–AcpPCI model. The presence of Dtx indicates Ddx-to-Dtx conversion that contributes to the activation of NPQ occurred preferentially in PSI–LHCI30, suggesting the role of PSI–LHCI in photoprotection.

Carotenoids play dual roles in the processes of excitation energy transfer: transferring excitation energy to Chls and quenching excessive excitation energy from Chls to facilitate photoprotection33–35. The AcpPCI-associated carotenoids have close interactions with Chls located within or around AcpPCIs (Supplementary Fig. 21k). This suggests that there is a highly efficient energy transfer between these pigment molecules. In addition, it is worth noting that Symbiodinium PSI–AcpPCI may possess multiple distinct energy quenching sites that are formed by the presence of previously unidentified Chls and Cars (Supplementary Fig. 21k–u). The previously unidentified Cars in PSI–AcpPCI (AcpPCI-5/Car410, AcpPCI-8/Car409/411/412, AcpPCI-9/Car 406/408/410, PsaI/Car 202, AcpPCI-1/Car 407/413) are in close proximity to Chls and are presumably responsible for dissipating excessive excitation energy from Chls (Supplementary Fig. 21k–q). The previously unidentified Chls (AcpPCI-9/Chl320, AcpPCI-1/Chl313/319, PsaB/Chl818, PsaL/Chl403/404/406/407/408, AcpPCI-3/Chl317/318, AcpPCI-7/Chl315) exhibit strong associations with Cars and have the potential to transfer excessive excitation energy to these Cars (Supplementary Fig. 21q–u). These unique structural features suggest that Symbiodinium PSI–AcpPCI may play a critical role in photoprotection.

Model of energy transfer within the PSI–AcpPCI supercomplex
Based on our structural data, we calculated excitation energy transfer (EET) within the PSI–AcpPCI complex. The EET rates between all Chl pairs were calculated using the Förster theory36. Moreover, the generalized Förster theory, an extension of the classical Förster theory, was employed to simulate the EET rates between AcpPCIs as well as between AcpPCIs and the PSI core37. The EET time constant between Chls, which is the reciprocal of the EET rate, reflects the dynamics of EET processes within the PSI–AcpPCI complex.

The EET time constant map for PSI–AcpPCI reveals that the EET rates between Chls occur on the picosecond timescale (Fig. 5a), consistent with the simulated EET rates of plant PSI–LHCI5. Many EET rates between Chls reach sub-picosecond rates, indicating rapid energy transfer within PSI–AcpPCI. The efficiency of EET within individual AcpPCIs and within the PSI core is higher compared to that between AcpPCIs and between AcpPCIs and the PSI core (Fig. 5a). Additionally, the stromal and lumenal Chl layers exhibit efficient EET (Fig. 5b). However, EET between AcpPCI-1 and AcpPCI-2, AcpPCI-2 and AcpPCI-12, AcpPCI-7 and AcpPCI-8, and AcpPCI-9 and AcpPCI-10 is less efficient due to the large gap between them (Fig. 5a, c). Consequently, AcpPCIs can be divided into three groups for energy transfer to the PSI core: AcpPCI-2/3/4/5/6/7/12/13 (Group I), AcpPCI-8/9 (Group II), and AcpPCI-10/11/1 (Group III) (Fig. 5a, c).

In Group I, the most efficient EET occurs from AcpPCI-3/5/7 to the PSI core (Fig. 5c). Notably, the EET rate from AcpPCI-7 to the PSI core is less than 1.0 ps, suggesting a rapid energy transfer pathway. Direct energy migration from AcpPCI-4/6 to the PSI core is less efficient, but their energy can be transferred at a high rate to AcpPCI-3 and AcpPCI-7, respectively. Thus, EET from AcpPCI-4/6 to the PSI core primarily occurs through the mediation of AcpPCI-3 and AcpPCI-7 (Supplementary Table 5). AcpPCI-12 and AcpPCI-13, being farther from the PSI core, transfer energy to the PSI core via AcpPCI-3 and AcpPCI-4. Intriguingly, AcpPCI-13 utilizes three distinct pathways for energy transfer to the PSI core with similar rates of ~12 ps (Supplementary Table 5). Direct EET from AcpPCI-2 to the PSI core is inefficient, but this appears to be compensated by the AcpPCI-2–AcpPCI-3–core pathway.

In Group II, AcpPCI-8 demonstrates a high-rate EET to the PSI core, while the EET from AcpPCI-9 to the PSI core is significantly slower due to the large gap between them (Fig. 5a, c). Instead, AcpPCI-9 efficiently transfers energy to the PSI core through the mediation of AcpPCI-8. In Group III, all three AcpPCIs efficiently transfer energy to the PSI core, with AcpPCI-1 (1.1 ps) and AcpPCI-10 (1.6 ps) exhibiting particularly rapid rates (Fig. 5c). Moreover, AcpPCI-11 possesses an additional efficient pathway for EET through AcpPCI-10.

It is worth noting that part of the EET map in PsaB is incomplete due to the absence of ten Chls (Figs. 4a, 5a). Energy from AcpPCI-8/9/10/11 is primarily transferred to P700 through the pathways located at the PsaI–PsaM side of PsaB. In contrast, the EET map in PsaA shows a denser arrangement despite also lacking ten Chls. The missing Chls in PsaA are situated in the marginal area and their absence is compensated by the presence of four Chls (AChl832-833, LChl407-408) (Fig. 4a). Furthermore, the interior Chls 11-14, absent in PsaB, are present in PsaA. These differences in the Chl association contribute to significant distinctions in the EET within PsaA and PsaB. Furthermore, P700 re-reduction kinetics revealed that the photochemical efficiency and electron transport rate of Symbiodinium PSI–AcpPCI are slightly higher than those of cryptophyte PSI–ACPI, suggesting that the reducing Chl content in the PSI core has no effect on energy transfer within Symbiodinium PSI (Supplementary Fig. 22).

The Chl 305/306 pair located at the stromal side is critical in mediating the EET from AcpPCI-2/6/8/9/10 to the PSI core, while Chls 311 and 312 mediate energy transfer from AcpPCI-5 and AcpPCI-4/7/11, respectively, to the PSI core at the lumenal side (Fig. 5d, Supplementary Table 6). Due to the orientation change of AcpPCI-1, its Chls 302/307/308 face the PSI core and transfer energy to the Chls 303/304/306 in PsaL. The pigments of AcpPCI-3/4 exhibit a conformational shift towards the PSI core compared to their counterparts in diatom FCPI-3/4 (Supplementary Fig. 23a, b). Among them, Chls 302-304 of AcpPCI-3 and Chls 308/312 of AcpPCI-4 form efficient EET pathways with the Chls A832 and A833, respectively. Additionally, Chl 318 of AcpPCI-3 transfers energy to Chls A824/A825 at a high rate, compensating for the loss of Chls A1, A3, and A5-A9 at the interface (Supplementary Fig. 23a, b). Chl 311 of AcpPCI-5, which is absent in FCPI-5, mediates efficient EET at the lumenal side to Chls A811/A813 of PsaA, compensating for the lack of EET pathways at the stromal side due to the absence of Chls 9/10 of PsaA (Supplementary Fig. 23c).

AcpPCI-1/3/4 exhibit changes in orientation compared to other AcpPCIs, resembling Lhcb9 in the PSI-LHCI supercomplex from moss Physcomitrium patens. In Lhcb9, the red Chl pair 603/609, which corresponds to the Chl pair 305/306 in AcpPCIs, is faced outward and is sandwiched by two carotenoids L2 and L3 (Supplementary Fig. 23h). This pigment arrangement was presumed to play a role in quenching excess energy in addition to light harvesting38,39. Intriguingly, a similar pigment arrangement was identified in AcpPCI-1, whereas the carotenoid equivalent to L3 in Lhcb9 is absent in AcpPCI-3/4 (Supplementary Fig. 23h). These findings suggest that AcpPCI-1 may function similarly to Lhcb9 in energy dissipating and light harvesting.

The previously unidentified Chl 315 in AcpPCI-7 plays a critical role in efficient EET pathways between AcpPCI-6/7 and the PSI core (Fig. 5d, Supplementary Fig. 23d). These pathways mediated by Chl 315 are absent in red algae, cryptophyte, and diatom PSI–LHCIs, indicating the efficient EET between AcpPCI-6/7 and the PSI core. The counterparts of diatom FCPI-7/Chl405 and FCPI-8/Chl409 are missing, reducing the lumenal energy transfer between AcpPCI-7 and 8 (Supplementary Fig. 23e). However, the Chl 314 in AcpPCI-7 at the stromal side and the Chl 316 in AcpPCI-8 at the lumenal side enhance energy transfer to the PSI core. Due to the absence of Chls B5-B7 in PsaB, energy from AcpPCI-8 is transferred to Chl R201 of PsaR through the Chl 305/306 pair and Chl 311 that is closer to Chl R201 compared to its diatom counterpart Chl 410 (Supplementary Fig. 23f). The reduced distance between Chl 311 and Chl R201 potentially promotes the energy transfer rate to compensate for the missing EET pathways resulted from the loss of Chls B5-B7. AcpPCI-8 lack the stromal Chl 413 present in FCPI-8, but it possesses the lumenal Chl 310 that is absent in FCPI-8, forming efficient EET pathways with the Chl 305/306 pair and Chl 309 of the shifted AcpPCI-9 (Supplementary Fig. 23f). The Chl 305/306 pair of AcpPCI-10 is closer to Chl B830 compared to those of FCPI-10, facilitating high-rate EET to Chl B830 (Supplementary Fig. 23g). The previously unidentified Chl B818 is positioned near AcpPCI-10/11 and accepts energy from the Chl 305/306 pair of AcpPCI-10 and Chl 307 of AcpPCI-11 (Supplementary Fig. 23g). Chl 312 of AcpPCI-11 which is absent in FCPI-11 transfers energy to Chl B812, thereby strengthening the EET between AcpPCI-11 and the PSI core (Supplementary Fig. 23g).

Chls 301/304/308/310/312 are the primary pigments responsible for energy transfer between AcpPCIs. However, due to the conformational shifts and rotations of AcpPCI-1/2/3/4/9/12/13 as well as the binding of previously unidentified Chls, various other Chls, such as Chls 302/303/305/306/307/309/311/315/319/320, also participate in EET between AcpPCI-1/2/3/4/7/9/12/13 and neighboring AcpPCIs (Fig. 5d, Supplementary Table 7). EET between the stromal and lumenal Chl layers of AcpPCI-4/5/6/7/8/10/11 is mainly facilitated by the Chl 305/306 pair at the stromal side and Chls 309/311/312 at the lumenal side. However, this pattern differs for AcpPCI-1/3/9/12/13, as Chls 311/312 are absent in AcpPCI-1/3/9/13 and Chl 311 exhibits a conformational shift in AcpPCI-12 (Supplementary Fig. 24). Instead, EET between the two Chl layers in AcpPCI-1 is mediated by the Chls 313/319, and in AcpPCI-3 by the Chls 317/318, the Chl 305/306 pair and Chls 303/309. EET between the two Chl layers in AcpPCI-9/12/13 is less efficient. However, due to the rotations of these AcpPCIs, their Chls form EET networks with Chls in the opposite layers of adjacent AcpPCIs (Supplementary Fig. 24). In addition, the Chl 315 in AcpPCI-7 form efficient EET pathways with Chl 305 in the opposite layer of AcpPCI-6.

Overall, the lack of specific Chls in the PSI core and the conservation of Chl-binding sites in AcpPCIs result in the loss of certain EET pathways that rely on those particular Chls. However, the emergence of previously unidentified Chls in the PSI core and AcpPCIs, along with the conformational rotations and shifts of AcpPCIs and Chls, leads to the formation of specific EET pathways in Symbiodinium PSI–AcpPCI.

Both dinoflagellates and diatoms are important primary producers in the ocean, although they have evolved distinct PSI–LHCI supercomplexes. Diatom PSI–FCPI possesses the most extensive antenna and pigment system, as well as the most complicated EET network, among all the previously reported PSI–LHCI supercomplexes. These features enable diatoms to effectively adapt to fluctuating light conditions. In contrast to diatom PSI–FCPI, Symbiodinium PSI–AcpPCI has reduced quantities of antennae and pigments in both the antennae and the PSI core. Nevertheless, the PSI core subunits and antennae of PSI–AcpPCI possess extended terminal domains that are absent in diatom PSI–FCPI. Furthermore, the antenna and pigment arrangements as well as the EET pathways in PSI–AcpPCI differ from those in diatom PSI–FCPI. These structural variations likely reflect the ability of dinoflagellates to adapt and survive in more stable light conditions and clearer water, as opposed to diatoms.

In summary, the cryo-EM structure of PSI–AcpPCI unraveled the architecture of the PSI–LHCI supercomplex in the symbiotic dinoflagellate Symbiodinium sp., which possesses specific PSI core subunits and antennae with extended terminal domains, as well as specific pigment arrangements for efficient energy transfer. These structural characteristics provide insights into the adaptive mechanisms of light harvesting and energy transfer employed by Symbiodinium PSI–AcpPCI, enabling them to thrive in their specialized environmental habitats.

Methods
Purification of the PSI–AcpPCI from Symbiodinium
Symbiodinium sp. GY-H50 (catalog number: GY-H50) was purchased from Shanghai Guangyu Biological Technology Co., Ltd. It was isolated by limiting dilution method from coral in Beibu Gulf (Guangxi province, China) and was monospecific. To verify the strain, we performed 18S ribosomal RNA and internal transcribed spacer (ITS) gene sequencing, confirming that the strain belongs to Symbiodinium. The ribosomal RNA gene sequences were deposited to the National Center for Biotechnology Information database with the accession numbers PP191135 and PP191136. Primers are listed in Supplementary Table 8.

The alga was grown in 8 L of F/2 medium under continuous light at 40 μmol photon m-2 s–1 at 23°C with bubbling of air. The cells at logarithmic phase were collected by centrifugation (6000×g, 10 min). The harvested cells were disrupted with glass beads in buffer A (25 mM MES-NaOH, pH 6.5, 1.0 M betaine, 10 mM MgCl2). The cell debris were removed by centrifugation (3000×g, 2 min). The thylakoid membranes in supernatant were collected by centrifugation at 21,000×g for 20 min, and then washed with buffer B (25 mM MES-NaOH, pH 6.5, 1.0 M betaine, 1.0 mM EDTA). The thylakoid membranes at 0.4 mg mL–1 Chl were solubilized with 2.8% (w/v) n-dodecyl-α-D-maltopyranoside (α-DDM) (Anatrace, USA) for 15 min in buffer C (25 mM MES, pH 6.5, 1.0 M betaine, 10 mM NaCl, 5.0 mM CaCl2) on ice in dark with gentle stirring. The solubilized membranes were centrifuged at 21,000g for 20 min at 4°C, and the supernatant was loaded onto a sucrose gradient of 10%-30% in buffer C containing 0.02% α-DDM. After centrifugation at 230,500g for 20 h (Beckman SW41 Ti rotor), the PSI–AcpPCI sample was collected in 20% layer, and then further purified by size-exclusion chromatography (GE; Superose 6 Increase 10/300 GL) in buffer D (25 mM MES, pH 6.5, 0.5 M betaine, 50 mM NaCl, 5.0 mM CaCl2). The sample was concentrated to 2.0 mg ml−1 in Chl using a 100 kDa cut-off filter (Amicon Ultra; Millipore) for cryo-EM specimen preparation. All steps were performed at 4°C under dim light.

Characterization of PSI–AcpPCI
Absorption spectra were recorded at room temperature with a Shimadzu UV–Vis 1990 spectrophotometer40. To analyze the subunit composition of PSI–AcpPCI, samples were separated by 8%-16% SDS-PAGE and identified by mass spectrometry analysis (Supplementary Data 1)13,41. The protein bands were cut out from the gel, reduced with dithiothreitol, alkylated with iodoacetamide, and digested with trypsin, and the resulting peptide fragments were examined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The peptides were separated with Easy-nLC 1000 System equipped a phase trap column (nanoViper C18, 100 μm × 2 cm, Thermo Fisher) connected to the C18-reversed phase analytical column (75 μm × 10 cm, 3 μm resin, Thermo Fisher) and coupled to a Q Exactive mass spectrometer (Thermo Fisher). The acquired spectra were searched against the selected database using MASCOT engine (version 2.4) with the ProteomeDiscovery searching algorithm (version 1.4).

Pigment composition of the PSI was analyzed by LC-20AD high performance liquid chromatograph (Shimadzu, Janpan) as described previously4. Pigments were extracted by pre-cooled 100% acetone overnight at 4°C protected from light. The extract was concentrated and injected into C18 reversed-phase column (Waters, Ireland). The elutes were detected by a Shimadzu photodiode array detector at 445 nm with a wavelength detection range of 300–800 nm. Seven pigments were identified based on the characteristic absorption peaks of their absorption spectra and elution profiles42, in line with previous reports43,44.

Sequence analysis of the PSI–AcpPCI from Symbiodinium
Transcriptome sequencing of Symbiodinium sp. were performed by BioMarker (BMK) through high-throughput sequencing. Total RNA was extracted from Symbiodinium cells, and the cDNA Library was constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, MA, USA) following the manufacturer’s recommendations. The first strand of cDNA was synthesized using the mRNA fragments as template with random hexamer primers and RNase H. DNA polymerase I and RNase H were used for synthesizing the second strand of cDNA. The synthesized double-stranded cDNA was modified by terminal repair, A-tailing, and adapter addition for hybridization. The cDNA fragments ∼150 bp in length were selected using the AMPure XP beads (Beckman Coulter, Beverly, USA). The cDNA was treated with USER Enzyme (NEB), and PCR was performed to obtain the final cDNA library. After clustering of the index-coded samples, the library preparations were sequenced on an Illumina HiSeq 2000 platform, and paired-end reads were generated. The transcriptome was assembled de novo based on the left.fq and right.fq using Trinity 2.5.145. Sequences of the PSI core and AcpPC subunits were identified by retrieving the homologous sequences in the National Center for Biotechnology Information databases from the transcriptome sequences. The sequence alignment was done using CLC Sequence Viewer 8.0 and ESPript 3.0.

Phylogenetic analysis
The sequences for producing phylogenetic trees were aligned with MUSCLE (default parameters), and the phylogenetic tree was constructed using MEGA X46. The evolutionary history was inferred using the Neighbor-Joining method47. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches48. The evolutionary distances were computed using the Poisson correction method49 and are in the units of the number of amino acid substitutions per site. All ambiguous positions were removed for each sequence pair (pairwise deletion option).

P700 redox kinetics
The P700 redox kinetics of cell suspensions at a Chl concentration of 0.24 mg ml−1 were measured with a Dual-PAM-100 (Walz) after dark adaptation. To measure the PSI photochemical efficiency, the slow kinetics of P700 were recorded. P700 was completely oxidized (Pm) by a 200 ms saturation pulse (SP) (maximum intensity at 625 nm, 10,000 µM photons m–2 s–1) after the cells were illuminated by far-red light at 720 nm (250 μM photons m–2 s–1) for 10 s, as SP induced multiple charge separations in the PSI reaction center. SP also induced multiple charge separations in the PSII reaction center and the resulting electrons were sufficient to fully reduce the secondary electron acceptor PQ. After SP is turned off, the oxidized P700 was fully reduced (Po) with great rapidity by electrons from PSII through PQ, cytochrome (Cyt) b6f, and plastocyanin. Forty seconds after the first SP, the actinic light (AL) (maximum intensity at 625 nm, 80 μM photons m–2 s–1) was opened, and a SP was applied every 20 s. The active and open PSI reaction center was completely oxidized (Pm’) by SP. The measurement was finished until the current P700 signal (P) and the Pm’ were stable. The photochemical efficiency of PSI was calculated: Y(I)=(Pm’–P)/(Pm–Po). The light curve was measured using the light curve program files, showing the relationship of the electron transport rate (ETR) and photosynthetically active radiation (PAR). The step width at each radiation intensity was 30 s.

Cryo-EM data collection and processing
Holey-carbon grids (Quantifoil Au R2/1, 200 mesh) were glow-discharged for cryo-EM sample preparation before 4.0 μL of PSI-AcpPCI sample (2.0 mg mL-1) was applied onto the freshly prepared grids. Then the grids were blotted in Vitrobot Mark IV (Thermo Fisher Scientific) working at 100% humidity at 8°C with a 1 s blotting time, force level of -1 and then were plunged into liquid ethane cooled by liquid nitrogen for vitrification. Data collection was performed on a 300 kV Titan Krios G3i microscope (Thermo Fisher Scientific) equipped with a K3 BioQuantum direct electron detector (Gatan Inc.) at a nominal magnification of ×81,000 corresponding to a pixel size of 0.53 Å on the specimen. The energy filter slit set to 20 eV (GIF, Gatan) and a total dose of 50 e-Å–2.

Image processing was mainly performed by cryoSPARC 3.3.151. All movie stacks were patch motion corrected and binned by a factor of 2 with dose weighting. The CTF parameters for each movie were estimated by Patch CTF Estimation before automatic particle picking. After particle-extraction and two rounds of reference-free 2D classification, 286,230 particles were selected, 167,360 particles were excluded from the 3D classification and 3D reconstruction and 3D classification. A final set with 118,810 particles were subjected to homogeneous refinement. To improve the resolution of the density map, after 3D non-uniform refinement and sharpening, global (per-group) CTF refinement, local (per-particle) CTF refinement and particle subtraction followed by local refinement with soft mask for the regions of the peripheral LHCIs (distinguished by 1, 2, 3 and 4) were performed using cryoSPARC. The overall resolutions of the map were 2.8 Å and the resolution of local maps were to 2.9 Å, 3.1 Å, 3.0 Å, and 3.2 Å based on the gold-standard FSC 0.143.

Model building and refinement
To build the model of Symbiodinium PSI–AcpPCI supercomplex, the PSI core and FCPIs in the innermost layer of diatom PSI–FCPI (PDB ID: 6LY5)4 were rigid body fitted into the 2.80 Å overall cryo-EM map and locally refined map using UCSF Chimera52. The amino acid residues of each chain were subsequently corrected by referring to the sequence of the counterpart in Symbiodinium from transcriptome sequencing. The previously unidentified subunit PsaT and PsaU and the additional terminal domains of the core subunits and AcpPCIs were constructed by de novo model building with COOT53. The potential sequences of AcpPCIs were searched from the transcriptome by blast using their homologous sequences from diatom (FCPIs). The potential sequences of PsaT and PsaU were searched from the transcriptome by blast using the estimated sequences of PsaT and PsaU based on their cryo-EM density maps (Supplementary Fig. 3). As the side chains of residues could not be identified precisely based on the cryo-EM density map at the current resolution, several sequences of PsaT and PsaU were estimated and each sequence is about 20 residues. Then, each of the potential sequences of PsaT, PsaU, and AcpPCIs was manually fitted into the density map, and amino acids with distinguishable side chains (such as phenylalanine, tyrosine, tryptophan, arginine, and glycine) were used to identify the proper sequence from the potential sequences (Supplementary Fig. 3). Chl a and Chl c were distinguished by the density map corresponding to the phytol chain for Chl a, and the planarity of C-181, C-18, C-17 and C-171 resulting from the C-18=C-17 double bound for Chl c3,13,54. Given the current resolution of the density map, it’s impossible to differentiate between diadinoxanthin and diatoxanthin. Therefore, the binding sites for both the putative diadinoxanthin and putative diatoxanthin were assigned as diadinoxanthin. The geometrical restraints of pigments were generated from the Grade Web Server, and all the residues and cofactors were manually adjusted with COOT. The constructed model was refined against the cryo-EM map by Phenix real-space refinement55. After real-space refinement, manual correction and adjustment was performed with COOT. Manual correction and Phenix refinement were carried out iteratively to improve the model quality. The geometries of the structural model were evaluated with Phenix and the statistics were summarized in Supplementary Table 1.

Simulation of the excitation energy transfer in PSI–AcpPCI
Excitation energy transfer within PSI–AcpPCI was studied in the limit of the Förster theory36. The energy transfer rate T (in ps−1) values were computed using the following equation,

T = 2π/ħ |Vmn|2 ∫ dω FD(ω) FA(ω) (1)

The electron coupling strength Vmn between pigments was calculated using the TrEsp method, which incorporates environmental screening factors56,57.

Vmn = f * ∑ l,l0 ql * ql0 / Rl,l0 (2)

f = { 1; R≤6.6
{ A*exp(-βR)+0.54; 6.6<R<20
{ 0.54; 20≤R (3)

Here, l refer to the l-th atom of pigment molecule m, qT denotes the transition charge of that atom, and R l,l0 represents the distance between the corresponding atoms in the pigment molecule. f is environmental screening factor.

In addition, the term FD(ω) refers to the emission spectrum of chromophore site m, while FA(ω) represents the absorption spectrum of chromophore site n. A Gaussian broadening function was employed to simulate the spectral properties of pigments, as demonstrated below58:

FD(ω) = 1/σm√(2π) * exp(-(ω-ωm)^2 / 2σm^2) (4)

FA(ω) = 1/σn√(2π) * exp(-(ω-ωn)^2 / 2σn^2) (5)

S is the Stokes shift, and σ represents the full width at half maximum (FWHM).

Furthermore, the generalized Förster theory, an extension of the classical Förster theory, was employed to simulate the energy transfer rates between LHCs as well as between LHC and PSI core pigment aggregates37:

k = 2π/ħ * ∑α,β exp(-εDα / kBT) / Z * |VDAαβ|2 * ∫ dω SD(ω) SA(ω) (6)

D refers to the donor aggregate, while A represents the acceptor aggregate. α and β denote the exciton states of the donor or acceptor aggregate, respectively. Specifically, α refers to the exciton state index of the donor aggregate, while β refers to the exciton state index of the acceptor aggregate. ε denotes the eigenvalue of the Hamiltonian for the donor or acceptor aggregate. Z is the partition function, given by Z= ∑α exp(-εDα / kBT). VDAαβ represents the electronic coupling between excitonic states of the donor and acceptor aggregates.

Gaussian 16 software (Gaussian, Inc. Wallingford, CT, USA) and custom Python scripts [https://doi.org/10.5281/zenodo.10791187]59 were used to calculate the above parameters. CAM-B3LYP/6-31G* was used and the keyword Iop(9/40=4) was added to the calculation to print out more detailed configuration coefficients for subsequent calculation of electronic coupling. When simulating the spectrum, the S and FWHM of Chl a were 160 cm-1 and 240 cm-160. In addition, the spectral overlap between Chl c and Chl a was replaced with experimental data.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Zhao et al. determine the single-parficle cryo-EM structure of the photosystem I-PCPI supercomplex that\nis found in a Synbiodinium species, which is a dinoflagellate that forms an endosymbiosis with corals.\nThe structure is compared primarily to similar PSI supercomplexes from red algae. The authors idenfify a\nnumber of interesfing characterisfics. First, the authors idenfify two new unexpected subunits bound to\nthe complex based on idenfifying their structure in the cryo-EM map. Second, the authors idenfify that\nsome looping regions provide novel interacfions due to them being much larger than those found in red\nalgae. Third, various chlorophyll sites are different between the dinoflagellate and red algal structures,\nwhich substanfially influences the energy transfer pathways. A highlight of the manuscript is the\ncalculafion and discussion of how energy is transferred through the chlorophyll sites, and how it is\ndifferent than red algae. This especially sets the manuscript apart compared to other manuscript that\nreport only structural features and speculate on energy transfer based solely on chromophore distances.\nI also note that the manuscript is very well wriften. I am especially impressed with the introducfion that\nnicely links the structural importance to the ecological context. The manuscript is thorough, fimely, and\nthe conclusions drawn are substanfiated. Thus, I enthusiasfically recommend the manuscript for\npublicafion pending some outstanding issues listed below.\nI ask that the authors provide more details on how the sequences of PsaT and PsaU were idenfified. I\nthink that at least a sentence or two should be added to the results secfion to clarify this. To my\nunderstanding, the authors idenfified certain amino acids based on the cryo-EM map, and then did a\nblastp to search for likely sequences. If not, please clarify. If so, please state which residues, and list\nsome similar sequences that are also candidates. It would be even more helpful if the authors showed a\nsupplementary figure showing the confidently assigned residues.\nLine 54: typo – … from a primordial endosymbiont.\nLine 469: I suggest replacing the word “junk” with “incorrectly selected parficle posifions” or something\nlike that. The word junk is probably not accessible to a broad audience.\nLine 469: Some numbers greater than 999 have commas (e.g., 118,810) and some do not (e.g., 286230).\nI suggest adding commas to numbers above 999.\nLine 536: The PDB code and the EMD code need to be switched so that they correspond correctly to the\nfirst part of the sentence.\nLine 716: In Figure 5, please add what the green shading means in panel a. Do they correspond to the\nthree groups I/II/III? If so, please change line 284 to 5a instead of 5c. The authors could also add labels\nfor each of the groups in 5a that would make the figure more accessible to the reader.", "reviewer_2": "The manuscript reports the characterizafion of the photosystem I complex (core + light-harvesfing\ncomplex) in a coral symbiont alga (Symbiodiniaceae) using the CryoEM technology. The composifion and\nstructural arrangement of the consfituent subunits are dissected using this powerful technique. The\nspeed of electron transfer between the components in the photosystem complex was also measured\nusing rapid light flash and fluorescence technology. As a non-CryoEM-expert, I am not enfirely qualified\nto evaluate how accurate the idenfificafion and organizafion modeling of the subunits are and how well-\nsupported the conclusions are. I marvel at the structural details of the photosystem that are revealed,\nhowever. I am more confident in commenfing on the biological implicafions of the findings.\nIt is very nice to know that the complex shares similarifies with its counterpart in diatoms. The absence\nand presence of subunits such as PsaK, PsaO, and PsaR across different lineages of algae are of interest.\nThe finding of PsaT and PsaU, which were previously unidenfified, and that they are also shared with\ndiatoms and some other red-family algae is parficularly interesfing. However, how the sequences of\nthese two subunits are determined is not so clear. Given that there is no prior knowledge (I assume that\nthey were not unknown in any photosynthefic organisms), how did the CryoEM informafion lead to the\nidenfificafion of the proteins from the large pool of proteins that are in these two subunits? Was it\nscreened by “try and error” one by one?\nMy more significant reservafion is with the limited funcfional insights that the findings seem to provide.\nWhich biological quesfion or which part of the unique biology of dinoflagellates or the specific species of\nSymbiodiniaceae can be explained by the findings? What advances in dinoflagellate biology or ecology\ndo these findings bring? Can they explain why this species can form a symbiofic relafionship with corals?\nI also have a few specific comments, which are given below.\nLines 95-98: there is no data for non-symbiofic Symbiodiniaceae or other dinoflagellates, this statement\nis not so appropriate. The same issue can be found elsewhere in the manuscript when the findings are\ncharacterized as “unique”.\nSupplementary Table 3 needs sources of informafion.\nMiss Chls: are those Chls lost due to sample processing or do they represent a physiological state?\nWhat are the “new” Chls? What is the “newly idenfified Chl xxx”?", "reviewer_3": "Symbiodinium, belonging to the dinoflagellates, plays a crucial role in the symbiofic relafionship with\ncoral, vitalizing reefs. However, environmental stress often triggers widespread coral bleaching events,\nseverely impacfing these biodiverse ecosystems. It has been reported that the Symbiodinium PSI–LHCI\nsupercomplex may be involved in the photoprotecfion process. In this manuscript, the authors describe\nthe cryo-electron microscopy structure of PSI-PCPI from Symbiodinium and exhibit disfincfive structural\nfeatures compared to their red lineage counterparts, including the novel subunits, sequence differences,\nconformafional changes and pigment distribufions. The results provide a solid structural basis for\nunraveling the mechanisms of efficient energy transfer pathways in PSI-PCPI supercomplexes and new\ninsights into the evolufionary diversity of PSI-LHC supercomplexes among different photosynthefic\norganisms. Overall, this is a very nice piece of work that advances the field. I have some concerns that\nthe authors should address before publicafion.\n1. About the PsaU subunit:\n1) Is the PsaU exclusive in dinoflagellate?\n2) Are the LPS1 in red algal PSI-LHC and PsaU in PSI-PCPI belong to homologs since they share a similar\noccupafion on the lumenal side of the PSI core?\n2. Line 137: The PsaI C-terminal extension may also cause a shortened N-terminus of PsaB.\n1) The author should also cite the Supplementary Fig. 7 to show the shortened N-terminus of PsaB.\n2) The shortened N-terminus should not be caused by the C-terminus of PsaI, and the word ‘cause’ could\nbe subsfituted by ‘adapt to’ or something else.\n3. Line 146 and Supplementary Fig. 12g: “The lumenal loops of PsaB (A215-S223 and G429-S449) are\nshortened, mirroring their 147 counterparts in PsaA (G213-L244 and A421-I431). This prevents\ninterference with the extended N148 terminus of PsaF and the lumenal loops of PsaR and PCPI-10\n(Supplementary Fig. 12g)”. The reducfions of the loops are not likely to belong to Symbiodinium PsaB\naccording to Supplementary Fig. 12g, while this sentence seems to assign the loops to Symbiodinium\nPsaB. It is confused, the author needs to make some correcfions and clarificafions.\n4. Line 142-143: ‘The extended N-terminus interacts with the N-terminus of PsaI….’ should be modified\nto ‘The extended N-terminus of PsaM interacts with the N-terminus of PsaI…’\n5. About PsaT (Lane 152-153): ‘PsaS subunit found in diatom PSI cores occupies a similar posifion to\nPsaT.’\n1) How about the sequence similarity between PsaS and PsaT?\n2) Are there any regularifies in the interacfion pafterns between PsaS/PsaT with the PSI core?\n6. Lane 234, ‘…loop region (G429-S449) result in the absence of B6 and B7 (Fig. 5e ...’ should be modified\nto Fig. 4e\n7. About PCPI-1 and PCPI-4:\nIt is reported that the Lhcb9 in Physcomitrium patens PSI-LHC supercomplexes adopts an orientafion\nopposite to the other LHCI monomers, its red Chl pair is faced outward and sandwiched by two\ncarotenoids, the pigments arrangement may play a role in quenching excess energy in addifion to light\nharvesfing (Sun, H et. al, Nature plants, 2023). It seems similar to the PCPI-1 and PCPI-4, which rotated\nby 180 and 90 degrees, respecfively. Does this pigment arrangement also exist in PCPI-1 or PCPI-4?\n8. About the material: Does the structure of the complexes change when algae transifion from a free-\nliving to a symbiofic lifestyle?\n9. Supplementary Table. 3/ PsaR also exist in red algal PSI-LHC (You et.al, Nature, 2023), the author\nshould revise it in this table."}, "author_response": "Responses to Reviewers’ Comments\nReviewer #1:\nZhao et al. determine the single-particle cryo-EM structure of the photosystem I-PCPI supercomplex that is\nfound in a Synbiodinium species, which is a dinoflagellate that forms an endosymbiosis with corals. The\nstructure is compared primarily to similar PSI supercomplexes from red algae. The authors identify a number\nof interesting characteristics. First, the authors identify two new unexpected subunits bound to the complex\nbased on identifying their structure in the cryo-EM map. Second, the authors identify that some looping regions\nprovide novel interactions due to them being much larger than those found in red algae. Third, various\nchlorophyll sites are different between the dinoflagellate and red algal structures, which substantially\ninfluences the energy transfer pathways. A highlight of the manuscript is the calculation and discussion of how\nenergy is transferred through the chlorophyll sites, and how it is different than red algae. This especially sets\nthe manuscript apart compared to other manuscript that report only structural features and speculate on energy\ntransfer based solely on chromophore distances. I also note that the manuscript is very well written. I am\nespecially impressed with the introduction that nicely links the structural importance to the ecological context.\nThe manuscript is thorough, timely, and the conclusions drawn are substantiated. Thus, I enthusiastically\nrecommend the manuscript for publication pending some outstanding issues listed below.\nReply: We sincerely appreciate Reviewer 1’s extremely positive comments on our work and manuscript.\n1. I ask that the authors provide more details on how the sequences of PsaT and PsaU were identified. I think\nthat at least a sentence or two should be added to the results section to clarify this. To my understanding, the\nauthors identified certain amino acids based on the cryo-EM map, and then did a blastp to search for likely\nsequences. If not, please clarify. If so, please state which residues, and list some similar sequences that are also\ncandidates. It would be even more helpful if the authors showed a supplementary figure showing the\nconfidently assigned residues.\nReply: Thanks for raising this important point and excellent suggestions. We have added additional panels\n(Supplementary Fig. 3b-3g) to clarify how we identified sequences of PsaT and PsaU. First, we selected\nsmall regions of the density maps of PsaT and PsaU with higher resolution (Supplementary Fig. 3b, 3e),\nand amino acid sequences were manually speculated based on the selected map (Speculation sequence)\n(Supplementary Fig. 3c, 3f). Then, blastp against the transcriptome sequences was performed using the\nspeculation sequences to identify potential sequences (Blastp sequence) (Supplementary Fig. 3c, 3f). Each\nof the potential sequences was manually fitted into the selected map. Amino acids with distinguishable side\nchains, such as phenylalanine, tyrosine, tryptophan, arginine, and glycine, were used to identify the proper\nsequence from the candidates (Supplementary Fig. 3b, 3e). The sequences were further confirmed by the\nsuitability of the side chains for the density map in other regions (Supplementary Fig. 3d, 3g). We have\nadded relevant descriptions in RESULTS AND DISCUSSION (lines 90-99) of the revised manuscript.\n2. Line 54: typo – … from a primordial endosymbiont.\nReply: Thanks for pointing this out. We have corrected the mistake.\n3. Line 469: I suggest replacing the word “junk” with “incorrectly selected particle positions” or something\nlike that. The word junk is probably not accessible to a broad audience.\nReply: We have revised the sentence as suggested by the reviewer.\n4. Line 469: Some numbers greater than 999 have commas (e.g., 118,810) and some do not (e.g., 286230). I\nsuggest adding commas to numbers above 999.\nReply: We have added commas to numbers above 999.\n5. Line 536: The PDB code and the EMD code need to be switched so that they correspond correctly to the\nfirst part of the sentence.\nReply: We have switched the PDB code and the EMD code.\n6. Line 716: In Figure 5, please add what the green shading means in panel a. Do they correspond to the three\ngroups I/II/III? If so, please change line 284 to 5a instead of 5c. The authors could also add labels for each of\nthe groups in 5a that would make the figure more accessible to the reader.\nrecolored Fig. 5a and 5c to indicate the Group I (green), Group II (blue), and Group III (orange) of PCPIs.\nWe have also added the color codes in figure legend and referred Fig. 5a in the corresponding sentence\n(lines 294-295).\nReviewer #2:\nThe manuscript reports the characterization of the photosystem I complex (core + light-harvesting complex)\nin a coral symbiont alga (Symbiodiniaceae) using the CryoEM technology. The composition and structural\narrangement of the constituent subunits are dissected using this powerful technique. The speed of electron\ntransfer between the components in the photosystem complex was also measured using rapid light flash and\nfluorescence technology. As a non-CryoEM-expert, I am not entirely qualified to evaluate how accurate the\nidentification and organization modeling of the subunits are and how well-supported the conclusions are. I\nmarvel at the structural details of the photosystem that are revealed, however. I am more confident in\ncommenting on the biological implications of the findings.\nIt is very nice to know that the complex shares similarities with its counterpart in diatoms. The absence and\npresence of subunits such as PsaK, PsaO, and PsaR across different lineages of algae are of interest. The finding\nof PsaT and PsaU, which were previously unidentified, and that they are also shared with diatoms and some\nother red-family algae is particularly interesting.\nReply: We sincerely appreciate Reviewer 2’s positive comments on our work.\n1. How the sequences of these two subunits are determined is not so clear. Given that there is no prior\nknowledge (I assume that they were not unknown in any photosynthetic organisms), how did the CryoEM\ninformation lead to the identification of the proteins from the large pool of proteins that are in these two\nsubunits? Was it screened by “try and error” one by one?\nReply: Please see our responses to the same question of Reviewer 1 above. We have added additional\npanels (Supplementary Fig 3b-3g) to clarify the determination of the sequences of PsaT and PsaU, and have\nadded relevant descriptions in RESULTS AND DISCUSSION of the revised manuscript.\n2. My more significant reservation is with the limited functional insights that the findings seem to provide.\nWhich biological question or which part of the unique biology of dinoflagellates or the specific species of\nSymbiodiniaceae can be explained by the findings? What advances in dinoflagellate biology or ecology do\nthese findings bring? Can they explain why this species can form a symbiotic relationship with corals?\nReply: Dinoflagellates are a prominent clade within the red lineage, and have ecological and evolutionary\nsignificance because they thrive as primary producers in diverse aquatic habitats and the plastids of\nphotosynthetic dinoflagellates originated from a primordial endosymbiont. However, despite studies on red\nalgae, cryptophytes, and diatoms, how Symbiodinium orchestrates the evolution of PSI–LHCI to drive\nefficient photosynthesis remains poorly understood.\nOur structural analysis, combined with computational calculation and spectroscopic analysis, reveals the\nstructural basis for distinctive protein organization, pigment arrangement, and excitation energy transfer\n(EET) pathways of Symbiodinium PSI–PCPI, which enable efficient light harvesting and energy transfer\nboth between antennas and from peripheral antennas to the PSI core. P700 re-reduction kinetics revealed\nthat Symbiodinium PSI–PCPI possesses high photochemical efficiency and electron transport rate.\nMoreover, to protect the photosynthetic apparatus from excess light in the shallow ocean, excitation energy\ncan be transferred directly from PSII to PSI (termed “spillover”) or dissipated as thermal energy (termed\nnonphotochemical quenching, NPQ) at P700+ in Symbiodinium (Gorbunov et al., Limnol Oceanogr, 2001,\n46: 75-85; Slavov et al., Biochim Biophys Acta, 2016, 1857: 840-847). Our structural data demonstrate a\nlarge amount of diadinoxanthin (Ddx) and diatoxanthin (Dtx) in Symbiodinium PSI–PCPI, and the\nconversion of Ddx to Dtx contributes to the activation of NPQ (Kato et al., Plant Physiol, 2020, 183: 1725-\n1734). In addition, the new Chl/Car-binding sites are located in the interface region between different PCPIs\nand between PCPIs and the PSI core, and form close interactions with surrounding pigments. These findings,\ntogether with the specific pigment organisation found in PCPI-1 (see our responses to Reviewer 3 below),\nindicate the important roles of PSI–PCPI in light harvesting and photoprotection. Our study elucidates the\nstructural basis for the efficient energy transfer and quenching in PSI–PCPI, which is critical for\nhosts, stabilizing the trophic foundation for symbiotic ecosystem in coral reefs.\nWe agree with both Reviewer 1 and Reviewer 3 that our study provides a solid structural basis for\nunravelling the mechanisms of efficient energy transfer pathways and energy quenching in the PSI-PCPI\nsupercomplex from Symbiodinium and provides new insights into the evolutionary diversity of PSI-LHC\nsupercomplexes among different photosynthetic organisms. We also agree that extensive spectroscopic\nanalysis of Symbiodinium PSI–PCPI remains to be investigated in our future studies to provide functional\ninsight into the special PSI–LHCI complex.\n3. Lines 95-98: there is no data for non-symbiotic Symbiodiniaceae or other dinoflagellates, this statement is\nnot so appropriate. The same issue can be found elsewhere in the manuscript when the findings are\ncharacterized as “unique”.\nReply: Thanks for the suggestion. We have replaced “unique” with “specific” or “novel” in the revised\nmanuscript. Despite the potential structural variations in species, our structural data do reveal remarkably\ndistinctive protein organization, pigment arrangement, and EET pathways of Symbiodinium PSI–LHCI,\nrelative to its red lineage counterparts (red algal PSI–LHCR, cryptophyte PSI–ACPI, and diatom PSI–\nFCPI). We agree that the structural details of PSI–LHCIs of non-symbiotic Symbiodiniaceae or other\ndinoflagellates remain to be characterised in future studies.\n4. Supplementary Table 3 needs sources of information.\nReply: Thanks for the suggestion. We have cited relevant references in Supplementary Table 3.\n5. Miss Chls: are those Chls lost due to sample processing or do they represent a physiological state?\nReply: We believe that the loss of Chls represents the physiological state of Symbiodinium PSI–LHCI. First,\nour sample processing was performed at 4 °C under dim light, and mild non-ionic detergent n-dodecyl-α-\nD-maltopyranoside (α-DDM) was used for membrane solubilization to reduce the disturbance of the sample.\nSecond, the absence of Chls in the Symbiodinium PSI core is ascribed to the conformational alterations of\napoproteins (Fig. 4, Supplementary Fig. 20). When compared to PSI–LHCI of other photoautotrophs, we\ndetermined distinct structural changes of apoproteins in Symbiodinium PSI–PCPI, including variations in\nloop lengths, conformational changes in the loop structures, and alternations in the residues surrounding\nChls. These alterations lead to insufficient space, lack of ligands, and reduction in hydrophobic and\nhydrogen-bond interactions for Chl binding. In summary, the loss of numerous Chls results in changes in\nthe pigment structure, which may contribute to the physiological adaptation to specific symbiotic\nenvironments.\n6. What are the “new” Chls? What is the “newly identified Chl xxx”?\nReply: Both “new” Chls and “newly identified Chl xxx” indicate the Chls that are unique to this\nSymbiodinium PSI–PCPI which are absent in other PSI–LHCIs. We have replaced “newly identified” with\n“new” to avoid confusion.\nReviewer #3:\nSymbiodinium, belonging to the dinoflagellates, plays a crucial role in the symbiotic relationship with coral,\nvitalizing reefs. However, environmental stress often triggers widespread coral bleaching events, severely\nimpacting these biodiverse ecosystems. It has been reported that the Symbiodinium PSI–LHCI supercomplex\nmay be involved in the photoprotection process. In this manuscript, the authors describe the cryo-electron\nmicroscopy structure of PSI-PCPI from Symbiodinium and exhibit distinctive structural features compared to\ntheir red lineage counterparts, including the novel subunits, sequence differences, conformational changes and\npigment distributions. The results provide a solid structural basis for unraveling the mechanisms of efficient\nenergy transfer pathways in PSI-PCPI supercomplexes and new insights into the evolutionary diversity of PSI-\nLHC supercomplexes among different photosynthetic organisms. Overall, this is a very nice piece of work that\nadvances the field. I have some concerns that the authors should address before publication.\nReply: We appreciate Reviewer 3’s highly positive comments on our work.\n1. About the PsaU subunit:\n3\n1) Is the PsaU exclusive in dinoflagellate?\nReply: We obtained homologous sequences of PsaU from the National Center for Biotechnology\nInformation databases. Our sequence alignment analysis showed that PsaU is exclusive in in marine\ndinoflagellates belonging to the genus Symbiodinium, with a high degree of conservation (Supplementary\nFig. 9i). This was stated in Page 5, line 124-125.\n2) Are the LPS1 in red algal PSI-LHC and PsaU in PSI-PCPI belong to homologs since they share a similar\noccupation on the lumenal side of the PSI core?\nReply: Thanks for the comment. Unfortunately, the amino acid sequence of L 1 in red algal PSI–LHC has\nPS\nnot been determined. Therefore, we were unable to perform the sequence comparation of L 1 and PsaU.\nPS\nHowever, the length and structure of L 1 are remarkably distinct from PsaU, although they share similar\nPS\nbinding positions within PSI–LHCI (Supplementary Fig. 8). We will analyse the sequence similarity of\nL 1 and PsaU in future once the sequence of L 1 is identified.\nPS PS\n2. Line 137: The PsaI C-terminal extension may also cause a shortened N-terminus of PsaB.\n1) The author should also cite the Supplementary Fig. 7 to show the shortened N-terminus of PsaB.\nReply: Thanks for the suggestion. As suggested by the reviewer, we have referred Supplementary Fig. 7 in\nthe corresponding sentence (lines 147-148) of the revised manuscript.\n2) The shortened N-terminus should not be caused by the C-terminus of PsaI, and the word ‘cause’ could be\nsubstituted by ‘adapt to’ or something else.\nReply: Thanks for pointing this out. We have replaced “cause” with “adapt to” as suggested.\n3. Line 146 and Supplementary Fig. 12g: “The lumenal loops of PsaB (A215-S223 and G429-S449) are\nshortened, mirroring their counterparts in PsaA (G213-L244 and A421-I431). This prevents interference with\nthe extended N terminus of PsaF and the lumenal loops of PsaR and PCPI-10 (Supplementary Fig. 12g)”. The\nreductions of the loops are not likely to belong to Symbiodinium PsaB according to Supplementary Fig. 12g,\nwhile this sentence seems to assign the loops to Symbiodinium PsaB. It is confused, the author needs to make\nsome corrections and clarifications.\nReply: As shown in Supplementary Fig. 12g, the PsaB subunits of Symbiodinium (red), red algae (gray),\ncryptophyte (orange), and diatom (blue) are superimposed to illustrate the contraction in length and the\nconformational changes of the lumenal loops of Symbiodinium PsaB (A215-S223 and G429-S449). We\nhave revised Supplementary Fig. 12g by adding labels and highlighting the loop regions with dashed oval\nfor better comprehension.\n4. Line 142-143: ‘The extended N-terminus interacts with the N-terminus of PsaI….’ should be modified to\n‘The extended N-terminus of PsaM interacts with the N-terminus of PsaI…’\nReply: We have clarified this sentence as suggested by the reviewer.\n5. About PsaT (Lane 152-153): ‘PsaS subunit found in diatom PSI cores occupies a similar position to\nPsaT.’\n1) How about the sequence similarity between PsaS and PsaT?\nReply: The sequence comparation of PsaS and PsaT can not be performed unfortunately, due to the fact\nthat the amino acid sequence of PsaS has not been determined in the diatom PSI-FCPI structure given its\nperipheral location (Xu et al., Nat Commun 2020, 11: 5081). Nevertheless, as shown in Fig. 2f and stated\nin our manuscript, the structures of PsaS and PsaT show a low similarity. We will analyse the sequence\nsimilarity of PsaS and PsaT in future once the sequence of PsaS is determined.\n2) Are there any regularities in the interaction patterns between PsaS/PsaT with the PSI core?\nReply: Although PsaS and PsaT locate at similar positions on the PSI core and have close interactions with\nPsaB/C/D/L, their detailed binding sites with PsaB/C/D/L are distinct given their low structural similarity\n(Fig. 2f). Moreover, PsaT also forms interactions with the extended C-terminus of Symbiodinium PsaI,\nwhich is absent for PsaS in diatom PSI.\n6. Line 234, ‘…loop region (G429-S449) result in the absence of B6 and B7 (Fig. 5e ...’ should be modified to\nFig. 4e\nReply: Thanks for pointing this out. We have corrected the typo in the revised manuscript.\n4\n7. About PCPI-1 and PCPI-4: It is reported that the Lhcb9 in Physcomitrium patens PSI-LHC supercomplexes\nadopts an orientation opposite to the other LHCI monomers, its red Chl pair is faced outward and sandwiched\nby two carotenoids, the pigments arrangement may play a role in quenching excess energy in addition to light\nharvesting (Sun, H et. al, Nature plants, 2023). It seems similar to the PCPI-1 and PCPI-4, which are rotated\nby 180 and 90 degrees, respectively. Does this pigment arrangement also exist in PCPI-1 or PCPI-4?\nReply: Thanks for the valuable comment. We have compared the pigment arrangement in Lhcb9 of\nPhyscomitrium patens (Pp) and those in Symbiodinium (Sy) PCPI-1, PCPI-3, and PCPI-4 which rotated by\n180, 180, and 90 degrees, respectively (Fig. R1 below, also added to Supplementary Figure 23). In Lhcb9,\nthe red Chl pair 603/609, which corresponds to the Chl pair 305/306 in PCPIs, is faced outward and is\nsandwiched by two carotenoids, L2 and L3. The Chl pair 305/306 and the carotenoid L2 are present in\nPCPI-1/3/4, whereas the carotenoid L3 exists only in PCPI-1. Therefore, the pigment arrangement of PCPI-\n1 is similar with that of Lhcb9, which is assumed to play a role in energy quenching in addition to light\nharvesting. We have added relevant descriptions in RESULTS AND DISCUSSION (lines 337-344) of the\nrevised manuscript.\nFig. R1. Comparison of the arrangement of the Chl pair 603/609 and carotenoids L2/L3 in Lhcb9 of\nmoss Physcomitrium patens (Pp) with those in Symbiodinium (Sy) PCPI-1, PCPI-3, and PCPI-4. The red\nChl pair 603/609 (corresponding to the Chl pair 305/306 in PCPI-1/3/4) and the two carotenoids L2/L3 of\nLhcb9 are labelled.\n8. About the material: Does the structure of the complexes change when algae transition from a free-\nliving to a symbiotic lifestyle?\nReply: Thanks for the great comment. We have now specified that the complex was obtained from\nSymbiodinium sp. in a free-living state in the revised manuscript (RESULTS AND DISCUSSION).\nPrevious studies reported the detection of numerous differentially expressed genes (DEGs) between the\nsymbiotic and free-living states of a coral symbiont genus (Yuyama et al., Microorganisms, 2021,\n9(8):1560). The results documented that the expression of genes related to photosynthesis was prone to\nincrease during endosymbiosis, suggesting an enhancement in photosynthetic activity in algae when they\nare in endosymbiosis with corals. This finding is consistent with previous work on endosymbiosis in\nSymbiodiniaceae, which indicated that the symbiotic environment promotes photosynthesis in the\nsymbiotic algae (Barott et al., PNAS, 2014, 112: 607-612). Our study characterised the structure of\nphotosystem I of Symbiodinium in its free-living state. Given that photosynthesis in symbiotic algae is\nenhanced during endosymbiosis, the structures of their photosystems may undergo structural changes. The\nconformational changes of photosystems as the symbiotic algae transition from a free-living to a symbiotic\nlifestyle remain a mystery and merits further investigations.\n9. Supplementary Table. 3/ PsaR also exist in red algal PSI-LHC (You et.al, Nature, 2023), the author\nshould revise it in this table.\nReply: Thanks for pointing this out. We have updated Supplementary Table 3 with additional information.\nReviewer #2 (Remarks to the Author):\nI am happy with the new revision, and I view the manuscript as being acceptable now.\n\nResponses to Reviewers’ Comments\nReviewer #1:\nThe authors have addressed all my comments appropriately and I am now happy to recommend the manuscript\nfor publication.\nReply: We sincerely appreciate the reviewer for endorsing publication.\nReviewer #3:\nThe author has revise their manuscript and answered my questions well. I recommend the manuscript for\npublication.\nReply: We sincerely appreciate the reviewer for endorsing publication.\nReviewer #2:\n1. The information of the isolate of Symbiodinium is missing: where was it isolated? How was it classified as\nSymbiodinium given the many genera that have been established for coral endosymbionts? How was the isolate\ndetermined to be monospecific? Where is the isolate? The strain must be deposited in a public algal culture\ncollection (e.g. NCMA) for public access for reproducing the work.\nReply: Thanks for raising this point. The strain used in this study, Symbiodinium sp. GY-H50, was purchased\nfrom the commercial algae bank, Shanghai Guangyu Biological Technology Co., Ltd.. Therefore, we could not\ndeposit it in a public algal culture collection. According to the information provided by the company (see the\nstrain certificate below), the species was isolated by limiting dilution method from coral in Beibu Gulf\n(Guangxi province, China) and was monospecific. It was identified as Symbiodinium based on 18S ribosomal\nRNA gene sequencing. To verify the strain, we have also performed 18S ribosomal RNA and internal\ntranscribed spacer (ITS) gene sequencing. confirming that the strain belongs to Symbiodinium. The ribosomal\nRNA gene sequences have been deposited to the National Center for Biotechnology Information database with\nthe accession numbers PP191135 and PP191136. We have now added the specification and Supplementary\nTable 8 in the revised manuscript.\nFig. R1. Certificate of the algal strain in this study\n2. Line 464: what wavelength of light was used in SP? Same question for subsequent steps of manipulation.\nReply: The maximum intensity of the light for saturation pulse (SP) and actinic light (AL) was 625 nm. We\nhave clarified this in METHODS.\n1\n3. Line 466: how was the “fully reduced with great rapidity” determined? Same question for lines 467-468.\nReply: A strong intensity of saturation pulse (SP) was used in this study (10,000 µM photons m-2 s-1). The 200\nms SP releases a significant amount of photons in a short time and induces multiple charge separations in the\nPSII reaction center. The resulting electrons are sufficient to fully reduce the secondary electron acceptor,\nplastoquinone (PQ). SP also induces multiple charge separations in the PSI reaction center, resulting in\ncomplete oxidation of P700 (Pm) (Fig. R2). After SP was turned off, the oxidized P700 was fully reduced with\ngreat rapidity by electrons from PSII through PQ, cytochrome (Cyt) b f, and plastocyanin (PC) (Fig. R2).\n4. Line 507: As PsaT and PsaU have not been discovered in any photosynthetic organisms, how come the\ndiatom homologs for use here as a reference?\nReply: We would like to clarify that we only used diatom homologs (FCPIs) for determing the PCPI sequences,\ninstead of PsaT and PsaU. To avoid the confusion, we have revised the corresponding descriptions in\nMETHODS (Page 19). “The potential sequences of PCPIs were searched from the transcriptome by blast using\ntheir homologous sequences from diatom (FCPIs). The potential sequences of PsaT and PsaU were searched\nfrom the transcriptome by blast using the estimated sequences of PsaT and PsaU based on their cryo-EM\ndensity maps (Supplementary Fig. 3). As the side chains of residues could not be identified precisely based on\nthe cryo-EM density map at the current resolution, several sequences of PsaT and PsaU were estimated and\neach sequence is about 20 residues. Then, each of the potential sequences of PsaT, PsaU, and PCPIs was\nmanually fitted into the density map, and amino acids with distinguishable side chains (such as phenylalanine,\ntyrosine, tryptophan, arginine, and glycine) were used to identify the proper sequence from the potential\nsequences (Supplementary Fig. 3).”.\n5. Lines 509-510: a question was raised in the first round of review, and it seems to stay unaddressed here:\nhow can the “best match” be defined without knowing the sequences in the first place?\nReply: Please see our responses for Q4 above. We have revised the corresponding descriptions and provided\ndetailed clarification in METHODS (Page 19).\n6. Lines 513-514: do you mean assign both putative diadinoxanthin and putative diatoxanthin as\ndiadinoxanthin?\nReply: We have revised the description in METHODS (Page 19). Given the current resolution of the density\nmap, it’s impossible to differentiate between diadinoxanthin and diatoxanthin. Therefore, the binding sites for\nboth the putative diadinoxanthin and putative diatoxanthin were assigned as diadinoxanthin.\n7. Line 519: with what criteria to terminate the iteration?\nReply: During the model refinement, we evaluated the geometries of the structural model after each round of\nmanual correction and Phenix refinement using the wwPDB Validation System and the validation program of\nPhenix. After several rounds of refinement, the model had no outliers in bond length, bond angle, chirality, and\nplanarity. As shown in the validation report, the Ramachandran outliers and sidechain outliers were 0.04% and\n0.12%, respectively, which were better than most of the EM structures. Based on these parameters, we believe\nthat the structural model in this study is qualified for publication.\n8. PsaT and PsaU peptide sequences should be made available for review now and for public access when\nthe paper is published.\nto the National Center for Biotechnology Information database with the accession numbers of PP196340 and\nPP196339, respectively. We have clarified this in the main text and Data Availability.\n9. Given the multiple Symbiodiniaceae genomes currently available, PsaT and PsaU homologs should be\nidentified and their potential evolutionary origin discussed.\nReply: Thanks for the suggestions. We have identified the homologous sequences of PsaT and PsaU. As stated\nin the manuscript, the homologous sequences are exclusive in Symbiodiniaceae and are only present in various\nspecies of Symbiodiniaceae. This suggests that PsaT and PsaU may play an important role in the adaptation\nand survival of Symbiodiniaceae in varying environmental conditions during evolution. We have added the\nphylogenetic analysis in Fig. R3 below and Supplementary Fig. 9j-k in the revised manuscript. Their potential\nevolutionary origin and physiological functions in the PSI-PCPI supercomplex merit further investigation.\nFig. R3. Phylogenetic tree of PsaT, PsaU and their homologous sequences. a, Phylogenetic tree of PsaT\nand its homologous sequences. b, Phylogenetic tree of PsaU and its homologous sequences. The neighbor-\njoining tree was based on amino acid sequences of LHCIs. The phylogenetic trees were built with the Poisson\nmodel, and a bootstrap test (1,000 replicates) was conducted.\n10. I find it unnecessary, and potentially confusing, to name the PCP in PSI as PCPI in the paper (including\nfigures), because the work does not involve PSII and PCPI gives incorrect impression as a different structure\nthan PCP. It is particularly confusing when PSI-PCPI is used, because it implies there is a PSI-PCPII etc.\nReply: We would like to emphasise that this is a general method approach for naming LHC that associate with\nPSI and PSII in photoautotrophic organisms. In green algae, moss, and land plants, the LHC complexes\nassociated with PSI were specifically named as LHCI and those associated with PSII were termed as LHCII\n(Qin et al., Science, 2015, 348:989-995; Qin et al., Nat Plants, 2019, 5:263-272; Gorski et al., Nat Plants,\n2022, 8:307-316; Wei et al., Nature, 2016, 534:69-74; Sheng et al., Nature Plants, 2019, 5:1320-1330). In\ndiatom, fucoxanthin-chlorophyll a/c proteins (FCPs) function as LHC, and the FCPs associated with PSI were\nnamed as FCPI, and those associated with PSII were termed FCPII (Xu et al., Nat Commun, 2020, 11:5081;\nPi et al., Science, 2019, 365:eaax4406). Additionally, in our previous study on the structure of cryptophyte\nPSI–LHCI, we named ACPI (alloxanthin-chlorophyll a/c protein) as the LHCI associated with PSI (Zhao et\nal., Plant Cell, 2023, 35:2449-2463). To keep the consistency in the field, we followed the conventional\nnomenclature and named the PCP in PSI as PCPI. We have specified this in the revised manuscript (Page 4).\n11. In Figure 5c, P1, P2, P3, …: do they stand for PCP1, PCP2, PCP3, …? Explain it in the legend.\nReply: We have added the clarification in the Figure 5 legend as suggested by the reviewer.\n12. Summarizing the findings with a schematic depicting the organization of the PSI and light harvesting\ncomplex components would be very meaningful.\nReply: Thanks for the suggestion. We have added the schematic models in Supplementary Fig. 14f to illustrate\nthe organizations of the PSI core and LHC components in Symbiodinium PSI–PCPI, red algae PSI–LHCR,\ncryptophyte PSI–ACPI, and diatom PSI–FCPI. We have added relevant discussion in the revised manuscript\n(Page 9).\n13. Related to the last comment, some comparative description of dinoflagellate PSI and diatom PSI would be\nmore meaningful than discussing the Symbiodinium PSI in the context of coral symbiosis. This is because both\ndinoflagellates and diatoms are important primary producers in the ocean but they adapt to different light\nfields: diatoms are more flexible to fluctuating light condition than dinoflagellates; diatoms are more adapted\nto turbid water whereas dinoflagellates prefer clear water, generally speaking.\nReply: Thanks for the great suggestions. In this study, we compared the structures of the PSI core subunits\nand LHCs, the arrangements of LHCs and pigments, and the EET pathways in Symbiodinium PSI–PCPI with\nthose of diatom PSI–FCPI, highlighting their remarkable differences. These structural variations between PSI–\nPCPI and PSI–FCPI could be attributed to the adaptations of dinoflagellates and diatoms to their respective\nenvironmental conditions. We have added relevant discussion in the revised manuscript (Page 14-15).\n14. References: refs 20 and 21 deal with quite different questions and the citation for the same context is a bit\nout of place. Anyway, the major conclusion of ref 21 has been contended and does not stand any longer (see\nhttps://doi.org/10.1078/1434461042650325). The authors are recommended to refer to up-to-date mainstream\nliterature on dinoflagellates.\nReply: Thanks for pointing this out. We have updated the literature on dinoflagellates.\n15. Finally, as the manuscript introduces the work with the mention that PSI is important for photoprotection,\nsome more discussion should be given as to how the observed PSI-PCP architecture confers a strong\nphotoprotective capacity. How the binding to dtx and ddx help NPQ? Through electron flow?\nReply: Carotenoids play dual roles in the processes of excitation energy transfer: transferring excitation energy\nto Chls and quenching excessive excitation energy from Chls for photoprotection. The PCPI-associated\ncarotenoids have close interactions with Chls located within or around PCPIs (Supplementary Fig. 21k). This\nsuggests that there is a highly efficient energy transfer between these pigment molecules. In addition, it is\nworth noting that Symbiodinium PSI–PCPI may possess multiple distinct energy quenching sites that are\nformed by the presence of new Chls and new Cars (Supplementary Fig. 21k-21t). The new Cars identified in\nPSI–PCPI are close to Chls and are presumably responsible for dissipating excessive excitation energy from\nChls (Supplementary Fig. 21k-21q). The newly identified Chls in PSI–PCPI exhibit strong associations with\nCars and have the potential to transfer excessive excitation energy to these Cars (Supplementary Fig. 21q-21t).\nWe have added additional panels (k-t) in Supplementary Fig. 21 to illustrate the potential energy quenching\nsites and have added relevant discussion in the revised manuscript (Page 10-11).\nThe Ddx-Dtx cycle converts the monoepoxide carotenoid, Ddx, into the de-epoxide form, Dtx, when\nexposed to intense light (Yamamoto et al., Meth Enzymol, 1985, 110: 303-312). Dtx dissipates excessive\nexcitation energy through fluorescence quenching and heat dissipation, resulting in significant NPQ (Demers\net al., Mar Ecol Progr Ser, 1991, 76:185-193; Olaizola and Yamamoto, Journal of Phycology, 1994, 30:606-\n612; Lavaud et al., Plant Physiol, 2002, 129:1398-1406; Goss et al., J Plant Physiol, 2006, 163:1008-1021).\n16. Related to the last comment, in line 118-120: how does this running of PsaI/J/L/M/R extended termini\nover the stromal surface of the PSI core provide a protection? Protection against light or what?\nReply: The extended termini of PsaI/J/L/M/R are located on the stromal surface of the PSI reaction centre,\nresulting in reduced interactions between the reaction centre and molecules in the stroma. This may mitigate\nthe damage of reactive oxygen species to the reaction centre and protect the pigments in the reaction centre\nunder high light. In addition, these extended termini form close interactions with the PSI core subunits and\nPCPIs, which may enhance the structural stability of PSI–PCPI under stress conditions. However, the function\nof these extended termini remains a mystery and merits further investigation. We have added relevant\ndiscussion in the revised manuscript (Page 5).\nReviewer #2 (Remarks to the Author):\nI am happy with the new revision, and I view the manuscript as being acceptable now."}]

Decision letter