Cryo-EM structure of a photosystem I supercomplex from Euglena gracilis containing pentameric LHCE and dimeric LHCII

Domain: neuroscience
Published: 2026-04-06
Editorial outcome: accept

Abstract
Euglenophyta is a representative phylum of green-lineage secondary endosymbiotic eukaryotes. These organisms have evolved far-red light-harvesting complexes (LHCs) composed of diadinoxanthin and chlorophyll a/b, which are now classified as the LHCE family. Here we report a 2.35-Å cryo-electron microscopy structure of photosystem I (PSI) supercomplex from Euglena gracilis, with all subunits and their paralogs assigned. This structure reveals a minimal PSI core associated with twelve LHCE and four LHCII subunits. Most LHCE subunits are organized into dimers through a helix C–to–helix C interaction. Two dimers, together with a monomeric LhcE8, assemble into a (2+2+1)-type LHCE pentamer. The red-shifted pairs in the two LhcE6 subunits likely contribute to far-red absorption. The LHCII subunits form a distinct heterodimer and associate with the PsaF side. Overall, these results provide a structural basis for understanding energy transfer and dissipation, antenna subunit assembly, and far-red light-harvesting strategies in green-lineage secondary endosymbiotic organisms.

Introduction
Eukaryotic photosystem I (PSI) is a membrane pigment–protein supercomplex composed of a reaction center surrounded by light-harvesting antenna complexes (LHCs). It functions as a highly efficient energy conversion apparatus, generating reducing power to drive the reduction of ferredoxin1. Cyanobacterial PSI is considered the evolutionary ancestor of all eukaryotic PSI supercomplexes2. Eukaryotic photosynthetic organisms, shaped by multiple endosymbiotic events, are broadly classified into the red and green lineages3,4.

In red algae and red-lineage secondary endosymbiotic organisms, PSI–LHCI supercomplexes exhibit substantial structural diversity in their LHC-binding sites5,6. In contrast, the LHC-binding sites in plants7-9 and green algae10-12, where four to ten LHCI subunits associate with the PSI core, are relatively conserved. The minimal PSI complex13, which lacks PsaG, PsaH, PsaK, PsaL, and PsaO, has been proposed as a structural "stepping stone" for the assembly of green PSI–LHCI supercomplexes14. Specifically, PsaG and PsaK are critical for binding the conserved LHCI belt15, whereas the PsaO, PsaL, and PsaH enable the association of phosphorylated LHCII with PSI during state transitions16-18. Nevertheless, high-resolution structures of PSI supercomplexes from green-lineage secondary endosymbiotic organisms remain unclear.

Euglena gracilis belongs to the Euglenophyta, a lineage derived from secondary endosymbiosis involving the engulfment of a green-lineage photosynthetic organism (a green alga)3,4, and utilizes conserved chlorophyll a/b for efficient energy transfer19. However, unlike typical green-lineage organisms, which employ the violaxanthin–antheraxanthin–zeaxanthin (VAZ) cycle for photoprotection20,21, E. gracilis has adopted the diadinoxanthin–diatoxanthin (Ddx–Dtx) cycle22; this cycle is commonly found in planktonic red-lineage chlorophyll a/c-containing marine algae21,23-25.

In addition, Euglena has developed a chlorophyll a far-red–absorbing antenna complex in E. gracilis–LHCE26, which is distinct from the Lhca and Lhcb proteins found in green plants, as well as Lhcf and Lhcr proteins in red-lineage species19. LHCE subunits have been shown to possess far-red light absorption capabilities. Five LHCE protein species (E1–E4 and E12) from the peripheral antenna pool are proposed to form a pentameric assembly, as suggested by low-resolution negative-stain EM data26. LhcE9 was suggested to be associated with PSII, and most LHCE subunits (E5–8, 10-13) are mainly distributed in PSI, along with 2–3 LhcbM proteins26.

In this work, to elucidate the high-resolution structure of PSI from a secondary endosymbiotic photosynthetic organism of green algal origin, and to understand the structural basis of its association with far-red–absorbing LHCE antenna supercomplexes, as well as their pigment network. Here, we purify the PSI supercomplex from Euglena gracilis (Eg) and determine its structure using single-particle cryo-electron microscopy (cryo-EM) at an overall resolution of 2.35 Å, with improved local resolution in the peripheral antenna regions achieved through seven rounds of local refinement. As a result, we model the Eg-PSI–LHCE–LHCII supercomplex, which comprises two LHCE pentamers, one LHCE dimer, one LHCII dimer, and two LHCII monomers. This structure provides a structural basis for understanding excitation energy transfer and dissipation, antenna subunit assembly, and far-red light-harvesting strategies in the PSI supercomplex of E. gracilis.

Results
Overall structure of the PSI–LHCE–LHCII supercomplex

We purified the PSI-LHCE-LHCII supercomplex from E. gracilis cells (GY-D32, Guangyu Biotech Co., Ltd., China) (Methods, Supplementary Fig. 1a) and confirmed the presence of LHCE and LHCII subunits through SDS-PAGE and mass spectrometry (Source data, Supplementary Fig. 1b). Under room temperature, the absorption spectrum of this supercomplex shows a Qy band peaking at 676 nm (Supplementary Fig. 1c), while the 77 K fluorescence emission spectrum peaks at 733 nm (Supplementary Fig. 1d)—approximately 20 nm red-shifted compared to typical green algal PSI18 but similar to plant PSI which contains red chlorophyll pairs7,9,27. This red shift corresponds to the red-shifted spectral properties of the abundant LHCE subunits incorporated within the PSI supercomplex26, and the emission spectrum shoulder at 686 nm corresponds to the LHCII subunits associated (Supplementary Fig. 1d). High-performance liquid chromatography (HPLC) analysis revealed the Eg-PSI-LHCE-LHCII supercomplex contains five major pigment species: chlorophyll a (Chl a), chlorophyll b (Chl b), diadinoxanthin (Ddx), neoxanthin (Neo) and β-carotene (Bcr) (Supplementary Fig. 1e).

The isolated Eg-PSI–LHCE–LHCII supercomplex was imaged by single-particle cryo-EM at 300 kV, and the data were processed using cryoSPARC28. Following Ab-initio reconstruction, 1,059,170 PSI particles were selected. A second round of 2D classification was then performed based on the heterogeneous refinement, and 51,871 particles were selected, which were subsequently refined to a resolution of 2.35 Å. To improve the local density in the peripheral antenna regions, seven rounds of masked focused refinement were carried out on distinct peripheral regions, resulting in reconstructions from 2.60 Å to 3.20 Å resolution (Supplementary Fig. 2 and 3). A composite cryo-EM map was generated from these refinements and used for model building and refinement (Supplementary Fig. 4 and 5; Supplementary Table 1; Methods). We built the initial model using ModelAngelo29 and CryoAtom30, as well as the recently reported full-length LHC sequences of E. gracilis26,31,32. We identified all subunits and their paralogs and performed manual model fitting, guided by sequence differences26,31and the local cryo-EM density.

The model of Eg-PSI–LHCE–LHCII supercomplex accommodates twelve LHCE subunits, comprising two LHCE pentamers and one LHCE dimer, four LHCII subunits, comprising one LhcbM dimer and two LhcbM monomers, and a minimal PSI core. LHCE pentamer 1 consists of LhcE5.4, LhcE5.7, LhcE6.2, LhcE7.3, and LhcE8.2; Homologous LHCE pentamer 2 consists of LhcE5.7, LhcE5.4, LhcE6.4, LhcE7.2, and LhcE8.2. The LhcE8 paralog in Pentamer 2 was tentatively assigned as LhcE8.2 based on local density fit and sequence homology to the corresponding subunit in Pentamer 1, as the resolution was insufficient to unambiguously distinguish between LhcE8 paralogs. Pentamer 1 and pentamer 2 are located on the PsaA and PsaB sides of the PSI core, respectively. The LHCE dimer is composed of LhcE10.2 and LhcE11.1 and is positioned at the interface between the PsaA and PsaJ subunits. The LHCII dimer comprises LhcbM2.1 and LhcbM8.2. The two LhcbM4 monomers are distributed separately, LhcbM4.6 associates directly with the PsaB side, facilitated by LhcbM2.1, while LhcbM4.10 is inserted between the two LHCE pentamers (Fig. 1a).

The minimal PSI core consists of only eight subunits: PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaJ, and PsaM (Fig. 1a, b; Supplementary Fig. 4a). Compared with the "stepping stone" minimal PSI13, the PSI core in E. gracilis employs an additional PsaM subunit and an extended PsaD (Supplementary Fig. 6). Notably, the extended PsaD and the additional PsaM mediate the association of LHCE pentamer 1 and LHCE pentamer 2 with the PSI core at the PsaA and PsaB sides, respectively (Fig. 1). The cryo-EM map also reveals 289 chlorophylls (Chls), 56 carotenoids (Cars) and 5 cofactors and 51 lipids, including 281 Chls a, 8 Chls b, 12 Bcrs, 42 Ddxs, and 2 Neos, 2 phylloquinones, 3 Fe S clusters, 3 digalactosyldiacylglycerol (DGDG) molecules; 21 monogalactosyl-diacylglycerol (MGDG) molecules; 23 phosphatidylglycerol (PG) molecules and 4 sulfoquinovosyldiacylglycerol (SQDG) molecules (Supplementary Table 2).

Structure and assembly of the peripheral LHCE subunits

Compared with typical green-lineage Lhca and Lhcb proteins7,33,34 and red-lineage FCPs23,35,36, all LHCE subunits identified in this study form a distinct evolutionary clade of antenna proteins (Supplementary Fig. 7). In detail, most LHCE subunits exhibit an extended AC-Loop (except LhcE8) and a shorter BC-Loop (except LhcE10) (Fig. 2a, Supplementary Fig. 8a).

All the LHCE subunits bind only two types of pigments: Chl a and Ddx. Each LHCE subunit combines two conserved Ddx-binding sites (Ddx616 and Ddx617) and eleven conserved Chl a-binding sites (Chl a601–604 and 608–614) (Supplementary Table 3). Notably, LhcE6 and LhcE7 coordinate an additional Chl a615 via an asparagine residue (Fig. 2a, Supplementary Fig. 8a, b). LhcE6–LhcE8 further coordinate an additional Chl a622 through a glutamate residue (Fig. 2a, Supplementary Fig. 8a, c). LhcE10.2 utilizes its extended BC-Loop to bind an extra Chl a621 (Fig. 2a, Supplementary Fig. 8a, d). As shown in Supplementary Fig. 7, phylogenetic analysis reveals that the LHCE subunits cluster into a distinct clade and share higher sequence similarity with adjacent Lhca proteins from green-lineage C. reinhardtii and plants than with red-lineage Lhcr proteins. This observation supports the evolutionary homology between LHCE and the light-harvesting complex family of green-lineage photosynthetic organisms. In addition, the chlorophyll-coordinating residues and carotenoid-binding sites of LHCE subunits also show homology to those in C. reinhardtii LHCI (Supplementary Fig. 9).

Most LHCE subunits employ the extended AC-Loop and utilize the Aspartic acid–Arginine–Serine (DRS) polar domain and tryptophan to form dimeric assemblies (Supplementary Fig. 8a, e). Three LHCE dimers: LhcE5–LhcE6, LhcE5–LhcE7, and LhcE10–LhcE11, were identified in this study (Fig. 2b). The LHCE dimer adopts a helix C–to–helix C interaction (Fig. 2b, Supplementary Fig. 8e), which differs from the helix A–to–helix C arrangement observed in green algae and plants7,9,10,33 but resembles the dimeric organization of red-lineage diatom FCPs23 (Supplementary Fig. 10a–c). In addition, the two conserved Ddx sites in LHCE subunits are similar to those of Ddx303 and Ddx305 in the dimeric FCP encoded by Lhcf735. In contrast, LhcE8.2 lacks the DRS polar domain and tryptophan therefore cannot form a stable dimer (Supplementary Fig. 8a, f).

Furthermore, two dimers: LhcE5–LhcE6 and LhcE5–LhcE7, along with a monomeric LhcE8, assemble into a (2+2+1)-type LHCE pentamer (Fig. 2c). This pentameric organization is mediated by the N-terminus of LhcE6 and LhcE8, which insert into the dimeric interfaces of the LhcE5–LhcE7 and LhcE5–LhcE6 dimers (Fig. 2c). The extended C-terminus of LhcE6 and LhcE8 are critical for pentamer stability (Fig. 2a, c; Supplementary Fig. 8a). Four MGDG and two PG lipid molecules are embedded at the subunit interfaces and strengthen the inter-subunit interactions (Fig. 2c). The LHCE pentamer resolved in this study differs from the previously reported (4+1)-type FCP pentamer in diatoms (FCPI tetramer and one monomer)37 (Supplementary Fig. 10d), and is also distinct from the hypothetical LHCE pentamer proposed under low-resolution conditions from negative stain26. In contrast, the LhcE10–LhcE11 dimer, due to its extended BC-Loop, sterically conflicts with adjacent subunits and therefore cannot participate in pentamer formation (Supplementary Fig. 8a, g).

The two homologous LHCE pentamers (P1 and P2) are located on the PsaA and PsaB sides of the PSI core, respectively (Fig. 1), with only minor structural differences arising from shifts in LhcE5.4 and LhcE5.7 (Supplementary Fig. 11). Notably, an extra Ddx618 is clearly resolved in LhcE8.2 of pentamer 1 (Supplementary Fig. 5a), where it is tightly bound at the interface between LhcbM4.10 and the LHCE pentamer. The density for Ddx618 is absent in pentamer 2, suggesting that it may be loosely bound in the peripheral region of the supercomplex.

Structure and assembly of the peripheral LHCII subunits

In plants and green algae, the PSI–LHCI supercomplex can associate with phosphorylated LHCII trimers during state transitions to form the PSI–LHCI–LHCII supercomplex16-18,38,39, thereby enabling the qT-type photoprotective mechanism40,41. In mosses, the PSI–LHCI–LHCII supercomplex has also been proposed to enhance overall light harvesting under low-light conditions42,43.

In this study, the Eg-PSI core employs a minimal structure and lacks several key subunits, including PsaO, PsaH, PsaL and PsaK, that are essential for LHCII binding in previously reported PSI–LHCI–LHCII complexes16-18,38. Despite this loss, we identify a previously unobserved LHCII heterodimer composed of LhcbM2.1 and LhcbM8.2. The monomer–monomer interaction within this heterodimer resembles that of the conserved LHCII trimer (Fig. 3a, b), and the heterodimer binds to the PsaF side of the PSI core (Fig. 1). PsaF has recently been suggested to function as a regulatory subunit that promotes the assembly of peripheral antenna complexes in PSI8.

In the heterodimer, both LhcbM2.1 and LhcbM8.2 coordinate an additional Chl a620 in their C-terminal regions (Fig. 3a, b). Compared with the LHCE subunits in this study, all Eg-LHCII subunits bind two conserved Car sites, 616, 617 and an extra 619 site. In the plant, mosses and green algal LHCII heterodimer, the conserved car619 site mostly modeled as neoxanthin34,42,44. In contrast, in this study, only two car619 sites in two LhcbM4 subunits could be modeled as Neo619 (Fig. 3c), based on the characteristic C29 chiral center (Supplementary Fig. 5a), two car619 sites in LHCII heterodimer are tentatively modeled as Ddx619 (Fig. 3a, b). Compared with plant and green algal LHCII subunits, which typically bind eight Chls a and six Chls b molecules34,44, only two coupled Chl b sites, Chl b606 and Chl b607, in the Eg-LHCII subunits could be tentatively identified (Fig. 3 and Supplementary Fig. 5b).

Interactions between antenna subunits and the PSI core

Interactions between antenna subunits and PSI core subunits were analyzed using van der Waals (VDW) contact analysis35 (Methods, Supplementary Fig. 12a). In addition, lipid molecules play crucial roles in stabilizing the oligomerization interfaces within the photosynthetic supercomplex45. We also characterized the distribution of lipid-binding sites in the PSI–LHCE–LHCII supercomplex, revealing their essential contributions to structural stability (Supplementary Fig. 12b).

In the complete PSI–LHCE–LHCII supercomplex, the two homologous LHCE pentamers (P1 and P2) are located on the PsaA and PsaB sides of the PSI core, respectively (Fig. 1 and 4a). LHCE pentamer 1 associates with the PSI core primarily through interactions between LhcE6.2 and LhcE7.3 of P1 and PsaA, facilitated by the extended N-terminus of PsaD (Supplementary Fig. 6). Six lipid molecules, inserted at the interface, strengthen these interactions (Fig. 4b), including SQDG 861, MGDG 624, 862, 863, 873, and PG 625.

For the LHCE pentamer 2 on the PsaB side, interactions between LhcE7.2 of P2 and PsaB on the stromal side are relatively weak. Instead, stable binding is mainly mediated by LhcE6.4 and LhcE7.2 of P2 interacting with PsaB and PsaM on the lumenal side. Two lipid molecules (SQDG856 and MGDG625) embedded at the lumenal interface strengthen the interactions (Fig. 4c). Between the two homologous LHCE pentamers, LhcbM4.10 is inserted into the interface between LHCE pentamer 1 and pentamer 2, strengthening their interactions with the assistance of three lipids: PG620, PG627, and SQDG623 (Fig. 4d).

The LhcE10–LhcE11 dimer associates directly with the PSI core through the extended BC-Loop of LhcE10.2 (Fig. 4e, Supplementary Fig. 8a), LhcE11.1 interacts with PsaA, facilitated by the lipid MGDG624 on the lumenal side (Fig. 4e). The LHCII heterodimer utilizes three lipid molecules (MGDG406, MGDG409, and SQDG407) to associate with PsaF and PsaJ of the PSI core (Fig. 4f). In addition, LhcbM4.6 forms interactions with the adjacent LhcbM2.1 subunit and binds to PsaB, facilitated by two lipid molecules, MGDG410 and MGDG852 (Fig. 4g).

Excitation energy transfer pathways within the LHCE pentamer and from peripheral antenna subunits to the PSI core

In this study, four Chl b606–Chl b607 pairs were assigned to the four LHCII subunits (Supplementary Table 3), as the additional carbonyl oxygen of each Chl b participates in a stable hydrogen-bonding network between the two pigments, producing a discernible density feature in the map (Supplementary Fig. 5b). Other potential Chl b sites in LhcbM subunits were tentatively modeled as Chl a, guided primarily by the higher overall abundance of Chl a indicated by HPLC results (Supplementary Fig. S1e). All chlorophyll-binding sites in the LHCE subunits are modeled as Chl a. The minimal PSI core, together with two LHCE pentamers, one LHCE dimer, one LHCII dimer, and two LHCII monomers, forms efficient excitation energy transfer (EET) pathways within the complete Eg-PSI–LHCE–LHCII supercomplex. We analyzed these EET pathways using Förster resonance energy transfer (FRET) analysis46,47 (Methods, Supplementary Fig. 13, Supplementary Table 4 and 5). The FRET parameters used in this study were derived from experimentally validated FRET constants obtained from previously reported spectroscopic models of Chls a and b in plant light-harvesting systems46,47. These parameters have been widely adopted in the field for modeling energy transfer in organisms containing Chls a and b, such as Chlamydomonas34 and moss43. The point–dipole approximation becomes unreliable for Mg–Mg distances below ~10 Å. Chlorophyll pairs within this range were excluded from quantitative FRET analysis due to potential inaccuracies in the model and their likely departure from the Förster regime. At such short distances, excitonic coupling and sub-picosecond energy transfer may occur, processes that are not adequately described by incoherent Förster theory48,49. To ensure the validity of our FRET analysis, our analysis is restricted to inter-subunit energy transfer pathways with Mg–Mg distances greater than 10 Å.

In the (2+2+1)-type LHCE pentamer of the complete supercomplex (Fig. 5a, b), EET pathways within the LHCE dimer are mediated by Chl a609–Chl a609 pairs, homologous with the Chl a406–Chl a406 pairs in diatom FCP23. EET pathways from dimer to dimer and from dimer to monomer are mediated by the Chl a615 in LhcE7 to Chl a613 in LhcE6, and from Chl a615 in LhcE6 to Chl a613 in LhcE8, respectively (Fig. 5b).

EET pathways from LHCE pentamer 1 to the PSI core are mainly mediated by Chl a614 →Chl a834 , Chl a612 →Chl a833 , Chl a611 →Chl a845 , and Chl a612 →Chl a824 (Fig. 5c). EET pathways from LHCE pentamer 2 to the PSI core are mainly mediated by coupled Chl a611-a612 pair →Chl a811-a812 pair (Fig. 5d). EET pathways between the two LHCE pentamers are mediated by Chl a608 in LhcE8.2 (P1) to Chl a601 in LhcE7.2 (P2), and EET pathways from LhcbM4.10 to the two pentamers are mainly mediated by Chl a609 →Chl a608 and Chl a601 , respectively (Fig. 5e).

EET pathways from the LHCE dimer to the PSI core are mainly mediated by the Chl a621 E10.2 , which is coordinated by its extended BC-Loop (Supplementary Fig. 8d), to Chl a815 , and by Chl a604 to Chl a816 (Fig. 5f). EET pathways from the LHCII dimer to the PSI core are mainly mediated by the Chl a620 (Fig. 3a, b), with the pathway proceeding as Chl a620 →Chl a620 →Chl a403 (Fig. 5g). EET pathways from LhcbM4.6 to the PSI core are mainly mediated by Chl a603 →Chl a841 and Chl a609 →Chl a822 (Fig. 5h).

We acknowledge the methodological limitations inherent in cryo-EM and Förster theory as previously described46,47. The cryo-EM data in this study represent a frozen snapshot (~90 K). Thermal flexibility at 300 K could alter pigment orientations and coupling. In addition, our reconstruction includes particles from different illumination or exposure times, and structural dynamics may blur subtle pigment arrangements. Even at 2.35 Å resolution, uncertainties of ±0.5 Å in Mg–Mg distances and ±10–20° in dipole orientation remain. Because EET rates scale with R⁻⁶ and κ², these uncertainties can alter computed rates by up to 50%. In this study, we do not address several known limitations of cryo-EM and related modeling methods—such as beam-induced motion, local resolution variability, radiation damage, and preferred particle orientation. In addition, the potential role of interfacial water molecules and surrounding electrolytes may also contribute to pigment stability and excitation energy transfer50-52. Because FRET rates depend sensitively on R⁻⁶, these experimental factors can introduce positional uncertainties of approximately 0.5 Å in the pigment coordinates53,54, potentially affecting the accuracy of energy transfer calculations. We acknowledged the methodological limitation. Previous studies about time-resolved fluorescence on PSI–LHCI revealed multiple kinetic components corresponding to distinct chlorophyll domains55,56, the current Förster model assumes a single homogeneous rate, which likely oversimplifies the dynamics. Limited quantitative validity of classical Förster modeling. The Förster-based kinetic model provides qualitative trends but not absolute rates. Without including site-energy disorder, reorganization energies, and coherence effects, the reported transfer constants should be interpreted as semi-quantitative.

Structural basis for red-shifted chlorophyll a absorption in Eg-PSI–LHCE–LHCII supercomplex.

In green-lineage PSI–LHCI supercomplexes, red-shifted absorption is typically contributed by red chlorophylls7,9. In land plants, the LHCI subunits (PLhca1-Lhca4-Lhca2-Lhca3) bind conserved red Chl a603–a609 pairs57, and the far-red shift is primarily caused by the Lhca3 and Lhca458-60. The identification of red chlorophylls should be based on multiple factors, including the axial ligands of the chlorophyll molecules and their local protein environment27. The variations in site energies of 100–200 cm⁻¹, induced by protein electrostatics, can strongly influence the formation of red-shifted states in PSI–LHCI complexes58,61,62.

Previous studies have shown that the substitution of His with Asn to Chl a603 can enhance far-red absorption 27,63, the replacement of Asn by His at this position (a603-NH substitution) results in the complete loss of far-red absorption and emission in both reconstituted Lhca3 (rLhca3) and rLhca4 complexes63. The 77 K fluorescence emission of canonical green-lineage PSI–LHCI supercomplexes varies across species: 734 nm in Arabidopsis thaliana27, 722 nm in Physcomitrium patens61, and 715 nm in Chlamydomonas reinhardtii33. These differences correlate with the axial ligand of Chl a603. In P. patens, Lhca3 uses Asn, but its Lhca4-homologous subunit Lhca2b retains His61, contributing to reduced red-shifting. In C. reinhardtii, both Lhca3 and Lhca8 use His for coordination27,33, explaining its weak far-red emission (Supplementary Fig. 14). In addition, the “large canopy”, defined as a cluster of bulky hydrophobic residues near the Chl a603–a609 pair, contributes to strongly red-shifted emission9,64,65. In Fittonia albivenis Lhca3, the sequence Phe-Trp-Phe-Gln forms an extended hydrophobic canopy that may lower the excited-state energy and supports 753 nm emission9. In contrast, A. thaliana, P. patens and C. reinhardtii Lhca3 contain the smaller Gly-Phe-Ile-Glu motif33 (Supplementary Fig. 15).

In this study, we found that the Chl a603–a609 pairs in the two LhcE6 subunits exhibit two key structural determinants of red-shifted chlorophylls: Asn-mediated ligation of Chl a603 and a distinct LhcE-specific canopy-like environment. Specifically, we performed simplified excitonic Hamiltonian calculations, which tentatively show that the Chl a603–a609 pairs may play an important role in the far-red spectral band splitting observed in LHCE subunits, as these pairs exhibit the largest excitonic energy splitting among all pigment pairs within each monomer (Supplementary Table 6). In addition, we found that the Chl a603–a609 pairs in LhcE6.2 and LhcE6.4 of pentamer 1 and pentamer 2 utilize Asn to coordinate with the Chl a603, in contrast to other LhcE subunits in this PSI supercomplex (Fig. 6a). On the other hand, each Chl a603–a609 pair in the LhcE dimer is surrounded by the LhcE-specific Arg-Met-Arg (RMR) domain (Fig. 6a) and the adjacent helix C (Fig. 6b), a feature arising from the helix C–to–helix C dimeric assembly of LHCE subunits (Fig. 2b), which creates a distinctive "canopy" effect (Fig. 6b). Furthermore, LhcE6.2 and LhcE6.4 in both pentamer 1 and pentamer 2 are located at the center of the LHCE pentamers (Fig. 6b), such that their Chl a603–a609 pairs are fully surrounded by adjacent LHCE subunits. This arrangement further enhances the canopy effect in LhcE6.2 and LhcE6.4. These structural features, which are associated with site energy and protein electrostatics, including Asn ligation, extended shielding by the RMR domain and adjacent helix C regions, and central positioning within the pentamer, provide potential insight into how a green alga-derived photosynthetic apparatus, following secondary endosymbiosis, evolved plant-like far-red light-harvesting capabilities, a feature likely attributable to the two LhcE6 subunits.

Insights into the evolution and antenna assembly mechanism of Eg-PSI supercomplex.

The overall architecture of the Eg-PSI–LHCE–LHCII supercomplex reveals an assembly mechanism distinct from those of previously characterized green-lineage PSI supercomplexes5,6. Compared to the canonical PSI–10LHCI supercomplex of C. reinhardtii33, the Euglena PSI–16LHCI complex binds more antenna subunits (Fig. 7). Notably, red-lineage secondary endosymbiotic algae extend their light-harvesting capacity by adding second- and third-layer LHCI antennas on the PsaA side5,25,66, a strategy that is derived from conserved antenna-binding sites in primary red-lineage phototrophs such as Porphyridium purpureum67, Galdieria sulphuraria68, and Cyanidioschyzon merolae69 (Fig. 7). In contrast, as a secondary green-lineage alga, Euglena employs an arrangement: the species-specific LHCE pentamer 1 and pentamer 2 form a second peripheral antenna layer that associates with both the PsaA and PsaB sides of the PSI core (Fig. 1). While antenna expansion on the PsaA side is common in other systems5, the PsaB-side expansion observed here is unprecedented among primary green-lineage plants and algae, as well as other photosynthetic eukaryotes5,6 (Fig. 7).

Notably, similar to PSI–LHCI supercomplexes in red-lineage secondary endosymbionts, no phosphorylation sites were identified at the LHCII–PSI core interface in this study, suggesting that the LHCII subunits are constitutively associated with the PSI core, thereby expanding the light-harvesting cross-sectional area. In red-lineage secondary endosymbiotic organisms, Lhcf and Lhcr family proteins are primarily associated with the PSII and PSI cores, respectively21,70. However, minor Lhcf subunits are constitutively bound to the PSI core and have been proposed to expand the light-harvesting capacity25,36,66,71, and minor Lhcr subunits associate constitutively with the PSII core and are suggested to stabilize antenna assembly35,72,73. This indicates that the association of PSII-related antenna subunits with the PSI core is not uncommon in secondary endosymbiotic organisms. In this context, the observation in Euglena, where LhcE9 is also predicted to associate with the PSII supercomplex26, fits a broader evolutionary pattern of flexible antenna allocation to optimize light-harvesting strategies during secondary endosymbiosis.

Methods
Purification of PSI-LHCE-LHCII from E. gracilis
PSI–LHCE–LHCII was purified from E. gracilis (GY-D32, Guangyu Biotech Co., Ltd., China). Cells were cultured in TAP medium18 under continuous illumination of ~100 μmol photons m-2 s-1, with 3% CO bubbling, at 25 ℃, on a 16-h light/8-h dark cycle under stirring at 120 rpm. All subsequent procedures were performed under green light and at 4 ℃ or on ice.

Cells were harvested by centrifugation at 4,000 g and resuspended in buffer containing 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-KOH (pH 7.5), 1 M betaine, 10 mM MgCl , and 5 mM CaCl . The cell suspension was disrupted using a French press (3 cycles at 30 MPa). Unbroken cells were removed by centrifugation at 1,000 g for 10 min. The supernatant was then centrifuged at 30,000 g for 30 min, and the resulting thylakoid pellet was resuspended in buffer containing 50 mM HEPES-KOH (pH 7.5), 1 M betaine, 10 mM NaCl, and 5 mM CaCl to a chlorophyll (a+b) concentration of 1 mg/mL.

The membranes were solubilized with 1.6% (w/v) n-dodecyl-α-D-maltoside (α-DDM) (Anatrace, Maumee, OH, USA) and incubated on ice for 20 min. Insoluble material was removed by centrifugation at 12,500 g for 10 min. The supernatant was loaded onto a continuous sucrose density gradient (0.1–1.3 M in buffer with 0.01% α-DDM) and centrifuged at 36,000 rpm for 18 h in a SW40 rotor (Beckman Coulter, USA). The PSI–LHCE–LHCII fraction was collected using a syringe (Supplementary Fig. 1a). Sucrose was removed by dilution with buffer (50 mM HEPES-KOH, pH 7.5, 110 mM NaCl, 5 mM CaCl , 0.03% α-DDM), and the sample was concentrated to 2.2 mg/mL total chlorophyll (a+b) using an ultrafiltration centrifuge tube (100 kDa molecular weight cutoff; Amicon, Merck Millipore, Germany).

Characterization of the PSI–LHCE–LHCII supercomplex
The subunit composition of the PSI–LHCE–LHCII supercomplex was analyzed by SDS-PAGE on a 12% polyacrylamide gel. The gel was stained with Coomassie Brilliant Blue R-250 (Sigma-Aldrich, Germany). Protein identification was performed by mass spectrometry. Briefly, Coomassie-stained bands were excised and subjected to in-gel digestion using sequencing-grade modified trypsin. The resulting peptides were extracted and analyzed by mass spectrometry (Source data).

Room-temperature absorption spectrum and fluorescence emission spectra at 77 K were recorded using a UV-Vis spectrophotometer (Shimadzu, Japan) and a fluorescence spectrophotometer (F-7000, Hitachi, Japan), respectively. Measurements were performed on the PSI–LHCE–LHCII supercomplex at a chlorophyll concentration of 50 μg Chl (a+b)/mL, with excitation at 436 nm.

Pigment composition was determined by HPLC42. Samples collected from the sucrose density gradient were mixed with 90% (v/v) acetone and incubated for 30 min at 4 ℃ to extract pigments. After centrifugation at 12,500 g for 15 min, the supernatant was loaded onto a C-18 column (5 μm, 100 Å, 250 mm × 4.6 mm; Grace, USA). Pigments were eluted at room temperature with a flow rate of 1 mL/min using a linear gradient from buffer A (90% acetonitrile in water) to buffer B (100% ethyl acetate). Pigment identities were assigned based on absorption spectra and retention times monitored at 445 nm. HPLC analysis revealed the presence of Chl a, Chl b, Neo, Ddx, and Bcr in the Eg-PSI–LHCE–LHCII supercomplex (Supplementary Fig. 1e). The Chl a/b ratio is about 10.6.

Phylogenetic analysis
To investigate the gene families of each antenna subunit, the sequences of the thirteen antenna proteins resolved in this study were compared with representative light-harvesting complex (LHC) proteins from green-lineage organisms, Arabidopsis thaliana27 and C. reinhardtii10, and the red-lineage diatom Thalassiosira pseudonana35,36. Multiple sequence alignment was performed using TBtools84, with protein sequences aligned using MAFFT85 under default settings. The resulting alignment was used to construct a phylogenetic tree in IQ-TREE286. The best-fitting substitution model was selected using the Bayesian Information Criterion implemented in IQ-TREE2, and the tree was visualized using iTOL v687.

Cryo‐EM data acquisition and processing
The PSI–LHCE–LHCII sample, at a concentration of 2.2 Chl (a+b) mg/mL, was applied to a Quantifoil Cu R1.2/1.3, 300-mesh grid and vitrified using a Vitrobot (Thermo Fisher Scientific, Mark IV, USA) at 4 ℃ and 100% humidity. Blotting was performed for 2 s with a blot force of 1.

Data were collected on a 300 kV Titan Krios microscope (Thermo Fisher Scientific) equipped with a Gatan Quantum energy filter (slit width: 20 eV) and a K3 Summit camera (Gatan, USA). Movies were acquired using EPU 1.9 software in super-resolution mode at a nominal magnification of ×130,000, yielding a calibrated pixel size of 0.92 Å. A total of 15,238 movies were recorded with a total dose of ~50.0 e⁻/Å², a dose rate of 20 e⁻/pixel/s (30 frames), and a defocus range of −1.0 to −2.0 μm. All micrographs were binned during processing, resulting in a final pixel size of 0.92 Å for downstream image processing.

Movies were imported into cryoSPARC28, and 3,935,064 particles were picked using the Blob/Template Picker. Particles were extracted and subjected to multiple rounds of two-dimensional (2D) classification to remove junk particles. A subset of 1,059,170 particles was selected for Ab-Initio reconstruction. Following two rounds of heterogeneous refinement, 51,871 particles were used to reconstruct the PSI–LHCE–LHCII supercomplex at a global resolution of 2.35 Å, as determined by the gold-standard Fourier shell correlation (FSC) at a cutoff of 0.143.

To improve the resolution of the peripheral antennae, local masks were made from 2.60 Å to 3.20 Å resolution, respectively. A composite map was generated by combining the global and local reconstructions and was used for model building and refinement (Supplementary Fig. 2).

Model building and refinement
For model building of the Eg-PSI–LHCE–LHCII supercomplex, the initial model building was assisted by ModelAngelo29 and CryoAtom30, followed by manual model fitting, guided by sequence differences26,31 and local cryo-EM density features (Supplementary Fig. 2 and 3). The assignments of the paralogs LhcE5, LhcE6, LhcE7, and LhcEbM4 were based on distinct structural features and clear differences in the cryo-EM density maps, including side-chain configurations and loop conformations (Supplementary Fig. 16 to 19). For LhcE8 in Pentamer 2, the local resolution is insufficient to unambiguously distinguish between closely related paralogs. Based on the best-fit density and sequence homology, this subunit was tentatively modeled as LhcE8.2.

Final model building and real-space refinement were performed using Coot 0.8.988. All protein residues and pigments were fitted using locally optimized map weights. Pigment assignment was according to the HPLC analysis (Supplementary Data Fig. 1e). Discrimination between Chl a and Chl b was based on the presence of the additional carbonyl oxygen atom of Chl b89. However, due to the limited resolution of the cryo-EM density, unambiguous identification of Chls b is tentative. During revision of our work, a 2.82 Å resolution structure of smaller E. gracilis PSI–13 LHCI was reported90. Due to low resolution in the local LHCI region, no Chl b molecules could be modeled in the structure. In contrast, in our study, four Chl b606-Chl b607 pairs in the four LHCII subunits (Supplementary Table 3) could be assigned, as the extra oxygen atom of each Chl b participates in a stable hydrogen-bonding network between the two pigments—providing a discernible density feature in the map (Supplementary Fig. 5b), other potential Chl b sites in LhcbM subunits are tentatively modeled as Chl a, guided primarily by the higher overall abundance of Chl a indicated by HPLC (Supplementary Fig. S1e).

Discrimination between Ddx and Bcr was based on the presence of bulged densities corresponding to the terminal oxygen atom (Supplementary Fig. 5a). Neo molecules could be distinguished based by its characteristic C29 chiral center (Supplementary Fig. 5a). In this study, only the density at the Car619 site in LhcbM4 clearly exhibits this bent conformation (Supplementary Fig. 5a), allowing us to assign it as Neo619. For other carotenoid sites in the antenna subunits, the densities were tentatively modeled as Ddxs, guided primarily by the higher overall abundance of Ddx indicated by HPLC (Supplementary Fig. 1e).

The Eg-PSI–LHCE–LHCII model was finally refined using real-space refinement in Phenix 1.2091. Model validation, rebuilding, and refinement were carried out iteratively using MolProbity 4.292, Coot 0.8.988, and Phenix 1.2091. Statistics for cryo-EM data collection, model building, and refinement are summarized in Supplementary Table 1. Figures were prepared using UCSF ChimeraX 1.1093.

VDW force analyses between PSI core and antenna subunits
VDW contact analysis was performed using a previously described method35. Parameters were set using the 'Contact' tool in UCSF ChimeraX 1.1093, and VDW interactions within individual antenna subunits and within the PSI core subunits were excluded. Only intermolecular VDW contacts between antenna subunits and the PSI core, and those mediated by lipid molecules are displayed (Supplementary Fig. 12).

Computational analysis of FRET
The equation of FRET is described as follows:
k = (C * κ²) / (n⁴ * R⁶) (1)
where C is the spectral overlap between the electron donor's fluorescence spectrum and the acceptor's absorption spectrum. The applied C values for Chl a→Chl a, Chl b→Chl a, Chl a→Chl b, and Chl b→Chl b were 32.26, 1.11, 9.61, and 14.45, respectively, which were estimated from plant PSI-LHCI and LHCII previously46,47.

The dipole orientation factor is defined as:
κ² = [𝐮̂_D ⋅𝐮̂_A - 3(𝐮̂_D ⋅𝐑̂_DA)(𝐮̂_A ⋅𝐑̂_DA)]² (2)
The 𝐮̂_D and 𝐮̂_A are the unit vectors of displacement vectors from NB to ND atom in donor and acceptor Chl, representing the direction of dipole moment in the dipole approximation of the Chl. 𝐑̂_DA is the unit vector of the displacement vector from the magnesium atom in donor Chl to the magnesium atom in acceptor Chl. n stands for the refractive index, and an estimated value of n = 1.55 was taken from previous studies. R_DA is the magnesium-to-magnesium distance between donor and acceptor Chls. The lifetime and half life is defined as:
τ = 1 / k_FRET, t_1/2 = ln2 / k_FRET (3)

Error Propagation in FRET
The FRET calculation is based on structural parameters extracted from the cryo-EM resolved pigment geometry. To incorporate finite accuracy of structure determination, we explicitly include two independent uncertainty sources:
Distance uncertainty: we treat the donor–acceptor separation R as having a fixed error of σ_R = 0.5 Å for all pairs.
Dipole-orientation uncertainty: we allow the donor and acceptor transition dipoles to deviate by ±20° from their nominal directions. Dipole uncertainty is projected onto an uncertainty in the orientation factor κ² through a statistical (Monte Carlo) procedure. Specifically, we model this ±20° angular uncertainty by uniformly sampling perturbed dipole directions within a cone of half-angle δ = 20∘ around each dipole. For each Monte Carlo draw i, we compute κ² from the standard Förster orientation factor. Repeating this procedure for N samples yields a distribution κ²_i (N = 50000). We define:
⟨κ²⟩ = (1/N) * Σκ²_i, σ_κ² = sqrt((1/N) * Σ(κ²_i - ⟨κ²⟩)² ) (4)
⟨κ²⟩ is used as the effective orientation factor in FRET calculation, and σ_κ² is carried as the uncertainty originating from the ±20° dipole-angle uncertainty.

Since FRET rate constant k ∝ κ²R_DA⁻⁶, the relative uncertainty is:
(σ_k / k)² = (σ_κ² / ⟨κ²⟩)² + (6 * σ_R / R_DA)² (5)
The absolute uncertainty of k is then:
σ_k = k * sqrt((σ_κ² / ⟨κ²⟩)² + (6 * σ_R / R_DA)² ) (6)
And the absolute uncertainties of τ and t_1/2 are given by：
σ_τ = τ * (σ_k / k) (7)
σ_t_1/2 = t_1/2 * (σ_k / k) (8)

Calculation of excitonic Hamitonian modeling of two Chls
For two coupled chlorophylls (donor D and acceptor A), the excitonic Hamiltonian is written as:
H = [[E_D, J], [J, E_A]] (9)
where E_D and E_A are the site energies (excitation energies) of the two Chls, and J is the electronic coupling94. We approximate J by a point-dipole interaction:
J = (1 / (4πε₀ε_r)) * [𝝁_D ⋅𝝁_A - 3(𝝁_D ⋅𝐑̂_DA)(𝝁_A ⋅𝐑̂_DA)] / R_DA³ (10)
We use the same NB to ND vector to approximate the dipole orientation and fixed transition dipole magnitudes taken from literature:
μ(Chl a) = 4.49, μ(Chl b) = 3.13 (11)
The eigenvalues of the Hamiltonian are:
E_± = (E_D + E_A) / 2 ± sqrt(((E_D - E_A) / 2)² + J²) (12)
If both donor and acceptor are Chl A (E_D = E_A = E₀):
E_± = E₀ ± |J| (13)
And the energy splitting under this condition is :
ΔE = 2|J| (14)

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "I have carefully evaluated the revised version of the manuscript in light of my detailed comments provided in the previous round. I am satisfied that the authors have thoroughly and constructively addressed major and minor points raised during the review process.\n\nIn particular, the revision demonstrates substantial improvement in the physical rigor and clarity of the work. The authors have strengthened the transfer analysis of excitation energy by explicitly addressing uncertainties related to pigment distances, dipole orientations, and the limitations of Förster-based modeling. Additional validation of the cryo-EM structure, including local-resolution analysis and conformational variability assessment, has significantly improved confidence in the structural interpretations. Importantly, the authors now clearly articulate the semi-quantitative nature of the kinetic estimates and appropriately contextualize their results within the existing PSI–LHCI/LHCE literature.\n\nThe manuscript has also benefited from expanded discussion of methodological limitations, spectral broadening effects, cryogenic versus physiological conditions, and comparative structural insights from recently published PSI supercomplexes. Overall, these revisions have resulted in a more balanced, transparent, and scientifically robust presentation.\n\nBased on the current version, I consider that my concerns have been adequately resolved. The manuscript now meets the standards of clarity, rigor, and completeness expected for publication in Nature Communications, and I therefore recommend acceptance.", "reviewer_2": "Feng et al. have carefully addressed the issues I have raised during initial reviewing process to my satisfaction, and those raised by two other reviewers as far as I can tell. I believe this revision is suitable for publication once they address two minor issues.\n\nIn lines 521-522. \"Due to limited resolution, its clear architecture was solved(26).\" I do not believe that this is a correct statement the authors imply. Perhaps, the following rephrasing is better: Due to limited resolution, only the overall shape of the pentamer was identified but without detailed information on the subunit arrangement in it.\n\nI would like the authors to move Supplementary Figure 16 into Figure 6 of the main text if Nature Communication permits.", "reviewer_3": "The authors have adequately addressed my comments, and the manuscript has been greatly improved. I have no further questions."}, "author_response": "We sincerely thank the reviewer for the thoughtful evaluation of our revised manuscript and for the constructive suggestions, which we have carefully addressed as follows:\n\nFirst, we have now revised the text \"Due to limited resolution, only the overall shape of the pentamer was resolved, and no detailed information on the subunit arrangement was obtained\" (Lines: 465–467).\n\nSecond, we gratefully acknowledge the reviewer’s valuable suggestion to elevate Supplementary Figure 16 into the main text to strengthen the presentation of our findings on antenna assembly. In line with this recommendation and to enhance the thematic coherence of the manuscript, we have reorganized the main figures as follows:\n- Parts of Supplementary Figures 14 and 15 have been incorporated into Figure 6 in the main text to better support the discussion of red-shifted chlorophyll pairs and their functional implications;\n- Supplementary Figure 16 has been promoted to Figure 7 in the main text, where it now illustrates key aspects of the antenna assembly mechanism.\n\nThis reorganization improves the logical flow of the figures and strengthens the narrative in both the 'antenna assembly' and 'far-red light harvesting' sections of the manuscript.\n\nWe thank the reviewer once again for the insightful feedback, which has greatly enhanced the clarity and impact of our manuscript."}]

Decision letter