Structural basis for the assembly and energy transfer between the cyanobacterial PSI core and the double-layered IsiA proteins

Domain: chemistry
Published: 2025-12-20
Editorial outcome: minor_revision

Abstract
Iron-limitation is a common stress factor in natural environments. To survive under iron-starved conditions, cyanobacteria overexpress iron stress-induced protein A (IsiA), which is crucial for light-harvesting and photoprotection. Multiple IsiA proteins form a single- or double-layered architecture encircling the photosystem I (PSI) core, forming various PSI-IsiA supercomplexes. The assembly and energy transfer mechanisms of double-layered PSI-IsiA supercomplexes remain unelucidated. Here, we present high-resolution structures of two PSI-IsiA supercomplexes isolated from the cyanobacterium Thermosynechococcus elongatus BP-1 cultured under iron-starved conditions. The PSI3-IsiA43 complex contains a trimeric PSI core surrounded by 43 IsiA subunits assembled into a closed double-ring. The PSI1-IsiA13 complex contains 13 IsiA proteins arranged in a double-layered architecture attached to the monomeric PSI core. Atomic force microscopy demonstrates the presence and distribution of different PSI-IsiA complexes within native thylakoid membranes isolated from iron-starved cells. Our findings provide insights into the structural variability and adaptive mechanisms of PSI-IsiA complexes.

Introduction
Cyanobacteria are among the earliest life forms on Earth, emerging approximately 2.4 billion years ago, and possess the ability to produce oxygen through oxygenic photosynthesis. Two essential components of this process are photosystems I and II (PSI and PSI), both of which are large membrane-embedded pigment-protein complexes, with their core complexes driving light-driven charge separation and electron transfer 1–4. These core complexes usually collaborate with their peripheral antennae, which contain a high concentration of pigments and are capable of absorbing photons across different wavelengths of light, thereby significantly enhancing the absorption cross-section of the photosynthetic units 5. Photosynthetic organisms adapted to different living environments have developed diverse peripheral antennae to efficiently capture solar energy 6–19. While most plants and algae employ membrane-embedded pigment-protein complexes associated with PSI and PSII cores as peripheral antennae, cyanobacteria utilize distinct antennae called phycobilisomes (PBS), which are large water-soluble pigment-protein complexes. These PBS are primarily located on the cytoplasmic side of PSII, where they harvest and transfer photon energy to the PSII core 20,21. In addition, certain cyanobacterial species possess membrane-embedded pigment-protein complexes, such as iron stress-induced protein A (IsiA), which functions as PSI antenna under stress conditions 22–24.

Iron availability is one of the main factors limiting the primary production of aquatic photosynthetic organisms, and iron limitation is a common stress constantly encountered by cyanobacteria living in natural aquatic environments 25. In response to iron stress, cyanobacteria induce the expression of IsiA and IsiB (flavodoxin, Fld) proteins to cope with stressful environments and ensure their normal growth 26–29. Flavodoxin, which binds to a flavin mononucleotide molecule, acts as a functional substitute for the PSI electron acceptor ferredoxin, compensating for the loss of ferredoxin due to iron deficiency 30. IsiA is homologous to the PSII core subunit CP43, which shares a similar architecture with six transmembrane helices; it is thus also termed CP43’. IsiA subunits can assemble into single-, double-, or multi-layered structures around the PSI core in vivo 31. IsiA binds 17 chlorophyll (Chl) molecules in total, and the spectroscopic properties of PSI-IsiA supercomplexes have demonstrated that IsiAs can efficiently absorb light energy and transfer it to the PSI core 23,31–33. These findings shed light on the intricate dynamics of light absorption and transfer within these complexes 34–37. In addition, IsiA proteins alone can self-assemble into IsiA oligomers, which have been suggested to play a role in energy quenching 31,33,38,39.

A few cryo-electron microscopy (cryo-EM) structures of various cyanobacterial PSI-IsiA complexes have been reported previously, including single-ring PSI3-IsiA18 complexes from the mesophilic cyanobacteria Synechocystis sp. PCC 6803 40 and Synechococcus elongatus PCC 7942 41, as well as from the thermophilic cyanobacterium Thermosynechococcus vulcanus (T. vulcanus) 42. In addition, the interaction of Fld with the PSI-IsiA supercomplex has recently been elucidated through the Synechococcus PSI-IsiA-Fld cryo-EM structure, revealing that three Fld proteins bind symmetrically to the trimeric PSI core 41. Recently, structural analysis of the PSI1-IsiA6 complex from Anabaena sp. PCC 7120 showed that a monomeric PSI core lacking the PsaL subunit binds to six IsiA proteins, with the C-terminal domain of one IsiA (IsiA2) folding in a manner similar to that of PsaL and occupying its position 43. Although substantial structural information is available for single-ring PSI-IsiA complexes, the nature of the double-ring PSI-IsiA assembly has not been characterized, and the precise mechanisms underlying the formation, function, and variability of cyanobacterial photosynthesis under iron-limiting stress remain unclear.

Here, we determine the structural organization and assembly of double-layered PSI-IsiA complexes using single-particle cryo-EM, atomic force microscopy (AFM), and complementary biochemical analyses. Our findings provide key insights into the structural and functional dynamics of cyanobacterial PSI-IsiA supercomplexes adapted to varying levels of iron stress.

Results
Iron-stress cell culture and sample characterization
The PSI-IsiA supercomplexes were purified from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (T. elongatus). To induce iron deficiency, we first cultured T. elongatus cells in the BG-11 medium containing standard Fe2+ concentration (0.021 mM) and then transferred the cells (Fe+ cells) into iron-deficient medium at two different dilution ratios (1/25 and 1/60, v/v). The different treatments resulted in two iron-limited culture conditions (moderate and severe), producing two types of cells, Fe− cell-1 and Fe− cell-2, respectively. Moreover, we further cultured Fe− cell-2 in iron-supplemented medium to obtain Fe−/+ cells. Our electrophoresis analysis of these cells demonstrated that IsiA accumulates in both Fe− cells, and its protein level increases with iron concentration decreasing (Supplementary Fig. 1a). In addition, we measured the absorption spectra of these cells (Supplementary Fig. 1b), and found that both Fe− cell-1 and Fe− cell-2 exhibit a blue shift in the Qy absorption band of chlorophyll a, with Fe− cell-2 exhibiting a more pronounced shift than Fe− cell-1 of approximately 7 nm compared to Fe+ cells. In contrast, Fe−/+ cells show a Qy absorption band of chlorophyll a at 681.5 nm and a greatly reduced IsiA accumulation, similar to Fe+ cells (Supplementary Fig. 1). These observations suggest that the IsiA protein level is directly related to the iron availability, and the Qy absorption peak in cyanobacterial cells could be used as an indicator of increased IsiA expression, consistent with the earlier reports linking IsiA accumulation to changes in spectral properties 31,32,44.

We then fractionated the solubilized thylakoid membranes from three cell types (Fe− cell-1, Fe− cell-2 and Fe−/+ cell) using sucrose density gradient centrifugation (Supplementary Fig. 2a) and characterized the major PSI fractions through negative staining electron microscopy. We found that the Fe− cell-1 culture conditions predominantly resulted in single-ring PSI-IsiA complexes, namely PSI3-IsiA18 (Supplementary Fig. 2b), whereas the Fe− cell-2 cultivation led to the formation of double-ring PSI-IsiA complexes, namely trimeric PSI3-IsiA43 and monomeric PSI1-IsiA13 (Supplementary Fig. 2c,d). In comparison, Fe−/+ cells primarily form the trimeric PSI core (PSI3) (Supplementary Fig. 2e), likely due to the reduction of IsiA proteins (Supplementary Fig. 1a). These findings suggest that the formation of either a single or a double ring of IsiA is primarily determined by the extent of iron limitation, and cyanobacterial PSI and PSI-IsiA complexes exhibit structural reversibility in response to the changes in iron concentration.

Next, we analyzed the protein composition and spectral properties of these PSI-IsiA complexes (Supplementary Fig. 3). Room-temperature absorption spectra showed that both the PSI3-IsiA18 and PSI3-IsiA43 complexes exhibited a Qy absorption band of chlorophyll a at 673 nm, whereas the PSI3 sample showed a peak at 679 nm (Supplementary Fig. 3b), similar to the spectral profiles of the Fe− and Fe+ cells (Supplementary Fig. 1b). Fluorescence emission kinetics measured via P700 oxidation demonstrated that the PSI3, PSI3-IsiA18, and PSI3-IsiA43 complexes possess increased P700 oxidation rates, implying that these PSI complexes exhibit increased light absorption cross-section (Supplementary Fig. 3c), in line with a previous report 32. HPLC analysis of both the PSI3-IsiA43 and PSI1-IsiA13 samples identified three major pigment peaks belonging to zeaxanthin (Zea), chlorophyll a (Chl a), and β-carotene (BCR) (Supplementary Fig. 3d), similar to the previously reported pigment composition of PSI3-IsiA18 41. Together, these findings suggest that multiple types of PSI-IsiA supercomplexes were formed in T. elongatus under different iron-deficient conditions, and that these IsiA proteins function as peripheral antennae of the PSI core with high efficiency in light-harvesting and energy transfer.

Architecture of PSI3-IsiA43 and PSI1-IsiA13 complexes
To gain further insights into the structural assembly of these double-layered PSI-IsiA complexes, we solved the cryo-EM structures of PSI3-IsiA43 and PSI1-IsiA13 at resolutions of 3.4 Å and 3.5 Å, respectively (Supplementary Figs. 4–6 and Supplementary Table 1). Our structural analysis showed that the PSI3-IsiA43 complex comprises a trimeric PSI core surrounded by a double-layered IsiA ring with overall dimensions of approximately 380×380×110 Å (Fig. 1a–d). The inner IsiA ring consists of 18 IsiA subunits arranged in the same three-fold symmetry as the trimeric core. We designated the IsiA subunit on the PsaK pole of one PSI core (Core-1, Fig. 1c) as IsiA-i-1 and the 18 inner IsiA proteins as IsiA-i-1 to IsiA-i-18 in an anticlockwise direction when viewed from the lumenal side. The architecture of this part closely resembles the previously reported structures of PSI3-IsiA18 40–42, with the IsiA ring exhibiting a slight rotational shift (Supplementary Fig. 7a). The outer ring consists of 25 IsiA subunits that asymmetrically bind to the outer face of the inner IsiA ring, designated IsiA-o-1 to IsiA-o-25, starting from the IsiA-i-1 pole. Consistent with previously reported IsiA structures 40–42, each IsiA protomer possesses 6 transmembrane helices and a short C-terminal helix located on the cytoplasmic side and coordinates 17 Chla and 4 carotenoid molecules. All 43 IsiA subunits share a similar orientation, with their short C-terminal helices extending toward the PSI core. This structural observation suggests that the specific orientation of IsiA subunits is critical for the well-ordered assembly of the entire complex.

The PSI1-IsiA13 structure consists of a monomeric core that lacks PsaL and binds 13 IsiA subunits arranged in two layers attached to the PSI core, with 6 copies in the inner layer and 7 copies in the outer layer (Fig. 1e). We also observed a density blob adjacent to the IsiA-o-7 protein, which presumably corresponds to another IsiA subunit, indicating that an additional IsiA subunit (IsiA-o-8) is also present in the PSI1-IsiA13 complex (marked by the red arrow in Supplementary Fig. 5b). However, the poor density suggests that this IsiA protein appears to be highly mobile, and thus, was not modeled into our structure. When superimposed with each of the three PSI cores in the PSI3-IsiA43 structure, PSI1-IsiA13 aligned well with only one PSI moiety (designated as PSI-moiety-1; Fig. 2a) containing Core-1, IsiA-i-1 to IsiA-i-6, and IsiA-o-1 to IsiA-o-7. In comparison, superimposition with the other two PSI cores showed that the outer IsiA subunits in the PSI1-IsiA13 structure exhibit a noticeable rotational shift of approximately 5° compared to the corresponding IsiA subunits of PSI3-IsiA43 (Fig. 2a and Supplementary Fig. 7b).

We next compared the PSI1-IsiA13 and the PSI-moiety-1 of the PSI3-IsiA43 complex to analyze their structural and compositional differences. We found that they exhibit similar subunit and cofactor compositions, except that the PsaL subunit is absent in the PSI1-IsiA13 complex, which was likely lost during our purification procedure due to its weak interactions with other core subunits. Despite these similarities, the structures of PSI1-IsiA13 and PSI-moiety-1 show notable differences (Fig. 2a and Supplementary Fig. 8). Compared to PSI3-IsiA43, the subunits IsiA-i-5~6 and IsiA-o-5~7 of PSI1-IsiA13 exhibited a noticeable shift (~8 Å) within the membrane toward the core, potentially hindering the incorporation of additional IsiA proteins. As a result, PSI1-IsiA13 maybe unable to form an intact PSI3-IsiA43 complex (Fig. 2a). However, it cannot be ruled out that the trimerization reshapes the monomeric conformation, ensuring the formation of complete IsiA rings. In contrast to the relatively flat transmembrane region of PSI3-IsiA43, the membrane-spanning region of PSI1-IsiA13 exhibited a more pronounced curvature (Fig. 2b and Supplementary Fig. 8). Specifically, compared to the corresponding subunit in PSI3-IsiA43, IsiA-i-1 and IsiA-o-1 in PSI1-IsiA13 shifted toward the lumen by 2-5 Å, whereas IsiA-i-6 and IsiA-o-7 moved toward the cytoplasm by 3-10 Å. These structural variations likely contribute to the differences in the binding modes and excitation-energy transfer (EET) pathways between PSI3-IsiA43 and PSI1-IsiA13 (see detailed analysis below).

Interactions between the IsiA inner and outer rings
Our structural analysis showed that adjacent IsiA subunits within the same ring are tightly associated, similar to those in previously reported PSI3-IsiA18 structures 40–42, and are crucial for maintaining the double-ring architecture. In contrast, IsiA proteins belonging to the inner and outer rings predominantly form relatively weak, nonspecific hydrophobic interactions (Supplementary Fig. 9).

Upon analyzing the PSI3-IsiA43 complex, we observed that the 18 IsiA molecules in the inner ring displayed three fold symmetry, whereas the 25 IsiA subunits in the outer ring exhibited an asymmetrical arrangement, resulting in different interaction patterns between different inner-outer IsiA pairs (Supplementary Fig. 9c–t). Despite this heterogeneity, the interactions between the inner and outer IsiA of each pair predominantly occur at two similar patches located on the cytoplasmic and lumenal sides (Supplementary Fig. 9a, b). In most cases, lumenal interactions involve Phe200, Pro199, and Leu197 on the outer face of inner IsiAs, and Chl504, BCR521, and Ile297 on the inner side of the outer IsiAs. The cytoplasmic interactions are contributed by Chl516, Trp235, and Leu238 from the outer face of the inner IsiAs, and by Trp28 and Trp29 on the inner face of the outer IsiAs. Moreover, Leu352 from the C-terminal helix of the adjacent IsiAs in the outer ring also contributes to cytoplasmic hydrophobic interactions. Furthermore, in four inner-outer IsiA pairs (i-2:o-1, i-4:o-4, i-7:o-8, i-15:o-19), a hydrogen bond is formed at the lumenal side between the side chain of Gln194 of IsiA-i and the side chain of Gln67 from IsiA-o (Supplementary Fig. 9d,f,i,q). In addition, a small number of specific hydrogen bond interactions were formed in other IsiA pairs, reinforcing the interactions between the inner and outer rings.

The core-IsiA and inner-outer IsiA interactions in the PSI1-IsiA13 complex differs slightly from those in PSI3-IsiA43, especially at the IsiA-i-6/IsiA-o-7 end, where the IsiA subunits are positioned closer to the core and exhibit vertical shifts (Supplementary Fig. 10). As a result, in the PSI1-IsiA13 structure, an extra hydrogen bond is formed between Glu349 from the C-terminal helix of IsiA-i-6 and Chl1218 of PsaB (Supplementary Fig. 10a), and additional hydrophobic interactions were observed between Trp28 of IsiA-i-5 and Phe310 and Phe311 of PsaB (Supplementary Fig. 10b). Overall, the IsiA-i-5 and IsiA-i-6 subunits of the inner ring bind more tightly to the PSI core than to those in the PSI3-IsiA43 supercomplex. Moreover, IsiA-o-7 also exhibits a tighter binding with IsiA-i-6, forming additional hydrophobic interactions and a hydrogen bond between Gln194 of IsiA-i-6 and Gln67 of IsiA-o-7 (Supplementary Fig. 10a). In addition to these horizontal movements within the membrane, IsiA-o-5, IsiA-o-6, and IsiA-o-7 in the PSI1-IsiA13 complex shift vertically, thereby altering their interaction surfaces with the inner layer. As a result, the associations within several inner IsiA-PsaB chlorophyll pairs differ from those observed in PSI3-IsiA43 (Supplementary Fig. 10c).

Rotation and assembly of the outer ring
Previous studies have shown that the single IsiA ring exhibits organizational heterogeneity, characterized by positional rotations of the IsiA subunits relative to the trimeric PSI core in the PSI3-IsiA18 complex 45. Here, we also investigated the heterogeneity of the IsiA layers in both the PSI3-IsiA43 and PSI1-IsiA13 complexes using multibody refinement in Relion 46 (Supplementary Fig. 11). We divided each of the two complexes into three bodies: the PSI core, inner IsiA layer, and outer IsiA layer (Supplementary Fig. 11a,c). When assessing PSI3-IsiA43, we found that the inner ring is stably associated with the PSI core, while the outer ring exhibits notable flexibility relative to both the core and the inner IsiA ring. Specifically, the outer ring shows a horizontal rotation of approximately 28.2 Å within the membrane plane but lacks vertical movement (Fig. 3a, b).

Similar to PSI3-IsiA43, we found that PSI1-IsiA13 also has a relatively stable inner IsiA layer and a flexible outer layer relative to the PSI core but exhibits more complex motion patterns compared to PSI3-IsiA43 (Fig. 3c, d). The outer IsiA layer exhibited simultaneous fluctuations in both horizontal and vertical directions. In particular, the outer IsiA subunits of PSI1-IsiA13 show a horizontal shift of ~16.7 Å and a vertical shift of ~10.0 Å. Together, these results indicate that the outer IsiA layer in the monomeric PSI1-IsiA13 complex undergoes more pronounced fluctuations than the corresponding part in the trimeric PSI3-IsiA43 complex.

Intriguingly, our negative-staining electron microscopy results showed that several different classes of PSI-IsiA complexes are present in the purified PSI3-IsiA43 sample (Fig. 4). These complexes exhibit variations in the number of IsiA proteins incorporated into their outer ring, shedding light on the potential assembly mechanism of the double-ring PSI-IsiA complex. Our results suggest that the trimeric PSI core may initially assemble with a single IsiA ring containing 18 IsiA subunits. Subsequently, additional IsiA proteins might bind to the outside of the inner IsiAs in a continuous manner, either by adding in one direction or extending in both directions, until a closed outer ring is formed (Fig. 4). Alternatively, the observed intermediates may reflect a native feature of incomplete PSI-IsiA complexes within the membranes, or could be a result of disassembly of larger complexes into smaller ones during purification.

Membrane organization of PSI-IsiA complexes
To examine the PSI-IsiA assemblies in a context resembling their native state, we visualized their structures and lateral arrangement in thylakoid membranes isolated from iron-starved T. elongatus cells (Fe− cell-2) using AFM (Fig. 5 and Supplementary Fig. 12). The trimeric PSI cores were clearly visible as high protrusions on the membrane surface (Fig. 5a, b). Moreover, we observed particles corresponding to the monomeric PSI core associated with double-layered IsiAs. The PSI3-IsiA43 and PSI1-IsiA13 structural models obtained from cryo-EM analysis were fitted nicely onto the AFM topograph (Fig. 5b and Supplementary Fig. 12), suggesting the cryo-EM structures likely reflect the natural physiological state of cyanobacterial PSI-IsiA under extreme iron deficiency.

Furthermore, we analyzed the distances between different PSI cores in adjacent PSI-IsiA supercomplexes, which have been demonstrated to be closely correlated with the number of IsiA rings in these supercomplexes 31. Our analysis showed that, in most cases, the distances between neighboring PSI core trimers corresponded to three, four, or five IsiA layers, with four IsiA layers being the dominant form. This analysis suggests that a significant proportion of the PSI-IsiA supercomplexes possess two layers of IsiA rings in Fe− cell-2 (Fig. 5c), and double-ring PSI-IsiA complexes appear to be prevalent in native thylakoid membranes from Fe− cell-2 (Supplementary Fig. 12). Together, these findings highlight the fact that PSI-IsiA complexes with varying structures exist in native thylakoid membranes, consistent with previous observations in Synechococcus elongatus PCC 7942 and Synechococcus elongatus UTEX 2973 31,44.

Pigment arrangement and potential energy transfer pathways
Our structures revealed the detailed arrangement of chlorophyll molecules in the PSI3-IsiA43 and PSI1-IsiA13 complexes, illustrating the sophisticated pigment networks in PSI-IsiA assemblies, particularly at the interface between the inner and outer IsiA rings. Notably, 731 Chl a molecules are present in the double IsiA rings of the PSI3-IsiA43 structure, which is approximately three times the number of core pigments. Of the total Chl a molecules in IsiA subunits, 473 are located on the cytoplasmic side and 258 on the lumenal side of the thylakoid membrane. In addition, the Chl molecules exhibit a continuous and uniform distribution on the cytoplasmic side, whereas they form clusters on the lumenal side (Supplementary Fig. 13). The uneven distribution of pigments between the cytoplasmic and lumenal sides of thylakoid membranes was also observed in previously reported PSI3-IsiA18 structures 40–42.

The dense arrangement of Chl molecules results in the formation of multiple interfacial Chl pairs, in which the two Chl molecules belong to two adjacent protein subunits and are located in close proximity (Fig. 6a, b and Supplementary Fig. 14). These Chl pairs, particularly those at the interface between the inner and outer IsiA subunits, may facilitate rapid EET within the PSI-IsiA complexes. Although the interaction patterns of different inner-outer IsiA pairs vary, our structures identify common Chl molecules potentially involved in efficient EET from outer to inner IsiA subunits, including Chl506, 516, and 517 on the outer face of inner-IsiA subunits, and Chl504, 508, 510, and 519 on the inner face of outer-IsiA subunits. Moreover, Chl519 of IsiA from the inner ring may also constitute the primary site for donating excitation energy to the PSI core, as elucidated in our previous report 41.

To estimate the EET efficiency, we next calculated the Förster resonance energy transfer (FRET) rates of interfacial Chl pairs in the two PSI-IsiA structures (Fig. 6c, d and Supplementary Tables 2, 3). According to the Förster theory, FRET rates strongly depend on the Mg-Mg distances and mutual dipole moment orientations between two adjacent chlorophylls. Although the limited resolutions (3.4–3.5 Å) of our structures do not allow a precise determination of these structural parameters, the position and orientation of each chlorophyll molecule in IsiAs are similar to those in previously reported PSI-IsiA structures 40–43 (Supplementary Fig. 15). Therefore, we suggest that while the calculated results may be biased regarding several individual EET pathways, the overall EET networks calculated based on our structures are largely reliable. Our analysis shows that the EET within the same IsiA ring/layer is highly efficient because of the presence of multiple rapid transfer pathways between two adjacent IsiA subunits. Moreover, in the PSI3-IsiA43 complex, the major EET pathways from outer to inner IsiAs are primarily achieved through the Chl pairs outer508-inner506, outer508-inner516, outer504-inner517, outer519-inner517, and outer510-inner516 (Supplementary Table 3), consistent with our structural observation. In addition, our data indicate that EET is highly effective on the luminal side, even with a smaller number of Chl molecules, in agreement with a previous report 40. Interestingly, we found that while efficient EET occurs in each inner-outer IsiA pair, the IsiA-i-6 in the trimeric complex may exhibit reduced efficiency in the transfer of excitation energy to the core (Fig. 6c and Supplementary Table 3). As a result, energy absorbed by IsiA-i-6 may first be transferred to neighboring IsiAs within the same ring, and then further transferred to the core. Furthermore, we calculated the FRET rates of interfacial Chl pairs using the PSI3-IsiA43 structure in which the outer IsiA ring undergoes a large-scale rotation in our multibody analysis (Fig. 3a, b and Supplementary Table 4). The results showed that the primary interfacial Chl pairs involved in the rapid outer-to-inner IsiA EET are similar to those in the original PSI3-IsiA43 complex, albeit belonging to distinct IsiA pairs (Supplementary Tables 3, 4). These analyses suggested that the rotational shift of the outer IsiA ring does not result in a significant change in the efficiency of outer-to-inner IsiA EET. This finding is consistent with previous results obtained for the PSI3-IsiA18 complex 45, showing that a robust EET from the IsiA antennae to the core occurs despite the large-scale rotation of the IsiA ring.

The inward shift toward the PSI core and vertical movement of the terminal IsiA subunits in the PSI1-IsiA13 supercomplex lead to slight differences in the potential energy transfer pathways compared to PSI3-IsiA43, particularly at the IsiA-i-6/IsiA-o-7 end (Fig. 6d). The closer association between IsiA-i-6 and the core enables Chl519 of IsiA-i-6 to transfer excitation energy to PsaB more effectively than the corresponding pathways in PSI3-IsiA43 (Fig. 6c, d and Supplementary Tables 2, 3). In contrast, EET between IsiA-o-7 and IsiA-i-6 in PSI3-IsiA43 is highly efficient, whereas the PSI1-IsiA13 complex shows fewer effective EET pathways (shorter than 40 ps) in the corresponding region (Fig. 6c, d), presumably due to the vertical shift of the outer IsiAs. Despite these differences, our structural and FRET analyses demonstrate that in various PSI-IsiA complexes, the excitation energy absorbed by IsiA proteins is rapidly equilibrated within the IsiA ring/layer and further transferred from the outer IsiAs to the inner IsiAs and the cores through multiple pathways. This is consistent with a recent study showing that multiple chlorophyll molecules in multiple IsiAs contribute together to the rapid energy transfer within PSI-IsiA complexes 36. The intricate energy transfer network observed in our structures enables effective and robust EET in both PSI-IsiA complexes, even under changing iron-limited conditions.

Methods
Induction, purification and characterization of PSI3-IsiA43 and PSI1-IsiA13 complexes
T. elongatus was cultured at 50 °C in BG-11 medium with continuous bubbling under white light at ~50 µmol photons m−2 s−1. The Fe+ cells were cultured in the BG-11 medium containing a standard concentration of Fe2+ (0.021 mM). To induce the accumulation of IsiA proteins, the Fe+ cells were transferred to the iron-free BG-11 medium at two different dilution ratios (1/25 and 1/60, v/v), resulting in Fe− cell-1 (moderate iron limitation) and Fe− cell-2 (severe limitation). The Fe− cell-2 was further cultured in iron-supplemented medium for five days to obtain the Fe−/+ cells (iron restored after severe limitation). The absorption spectra of cells grown under iron-deficient conditions were measured to monitor IsiA protein accumulation. Cells were harvested by centrifugation at 5000 × g for 5 min, when the Qy absorption peak shifted from 683 to 676 nm. To obtain thylakoid membranes, the cell pellet was resuspended in a buffer containing 20 mM HEPES, pH 7.5, 20 mM NaCl, 10 mM CaCl2, 10 mM MgCl2 and disrupted using a high-pressure homogenizer at 750 bar. Unbroken cells were pelleted by centrifugation at 2500 × g for 5 min, and the resulting supernatant was subjected to ultracentrifugation at 70,000 × g for 40 min to collect the thylakoid membranes 41.

To purify the PSI3-IsiA43 and PSI1-IsiA13 complexes, thylakoid membranes were resuspended in 20 mM HEPES (pH 7.5), 10 mM CaCl2, and 10 mM MgCl2 at 0.7 mg Chl per ml. The membranes were then solubilized with 1% (w/v) dodecyl-β-d-maltoside (β-DDM, Anatrace) for 20 min on ice. The solubilized membranes were then loaded onto the 0.1–1.5 M sucrose density gradient in a buffer containing 20 mM HEPES pH 7.5, 10 mM CaCl2 and 10 mM MgCl2 and 0.03% (w/v) dodecyl-α-d-maltoside (α-DDM, Anatrace), following centrifugation at 154,390 × g for 19 h using a SW41 rotor (Beckman) at 4 °C. The PSI3-IsiA43 and PSI1-IsiA13 complexes were concentrated using Amicon Ultra Centrifugal Filters (Millipore) with 100 kDa cut-off, to final concentrations of 3 and 2 mg ml−1 (in Chl), respectively, for cryo-EM specimen preparation.

The room-temperature absorption spectra of T. elongatus cells, PSI and PSI-IsiA complexes were measured using a spectrophotometer (Hitachi, Japan). Pigment composition was analyzed using an HPLC system (Shimadzu, Japan). Samples were mixed with cold acetone (final concentration of 80% (v/v)). The mixture was vortexed and then centrifuged at 13,000 g for 10 min to extract pigments. The resulting supernatant was injected to a C-18 reversed-phase column (Shim-pack GIST C18, Shimadzu), eluted at a flow rate of 1 mL/min at room temperature, and monitored at 440 nm. The elution profile was as follows: From 0 to 1 min, 100% buffer A containing 87% (v/v) acetonitrile and 10% (v/v) methanol was used. From 1 to 20 min, a linear gradient of buffer A decreasing from 100 to 0% and buffer B (Methanol: Hexane = 8:2) increasing from 0% to 100% was applied. From 20 to 23 min, 100% buffer B was used. From 23 to 24 min, a linear gradient of buffer B was applied, decreasing from 100% to 0%. Finally, from 24 to 28 min, 100% buffer A was restored. Individual pigments were identified by the absorption spectrum of each elution peak.

P700 oxidation was measured as a transient decrease in absorption at 705 nm using a Joliot-type spectrophotometer (Bio-Logic SAS JTS-10). All samples were diluted into the same chlorophyll concentration, and 3 mM sodium ascorbate (electron donor) and 1 mM methyl-viologen (electron acceptor) were added to each sample. Two actinic lights of 630 nm (10 nm FWHM, 20 uE) and 720 nm (10 nm FWHM, 400 uE) were used for the measurement. The PSI oxidation kinetics were fitted with a mono-exponential model.

The protein composition of T. elongatus cells, the purified PSI3-IsiA43 and PSI1-IsiA13 complexes were analyzed using 4–20% (w/v) gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gel was stained with Coomassie Brilliant Blue R250. The band potentially corresponding to the IsiA protein was excised and digested with trypsin overnight. The digested sample was analyzed on a nanoLC Q-Exactive mass spectrometer (Thermo Fisher Scientific) in line with an Easy-nLC 1000 HPLC system (Thermo Fisher). MS/MS spectra from each LC-MS/MS run were searched against the Thermosynechococcus elongatus BP-1 sequence using the Proteome Discoverer (Thermo Fischer Scientific version 1.4.0.288) searching algorithm with the following criteria: Full tryptic specificity was required, two missed cleavages were allowed, precursor ion mass tolerance was set to 10 ppm, fragment ion mass tolerance was 0.02 Da, and peptide FDR (false discovery rate) was set to 1%.

Grid preparation and data acquisition
Aliquots (3 μL) of the sample (PSI3-IsiA43 or PSI1-IsiA13) were placed onto a freshly glow-discharged holey carbon grid (GIG-C311) and blotted for 4 s at a blotting force of level 3 using Vitrobot Mark IV (Thermo Fisher). The grids were then vitrified by plunging into liquid ethane and stored in liquid nitrogen.

PSI3-IsiA43 grids were imaged using a 300 kV Titan Krios microscope (Thermo Fisher) equipped with a K2 direct electron detector (Gatan). Movies (32 frames per movie file) were captured at a defocus range of −1.5 to −2.5 μm at 130,000 × magnification in super-resolution mode. The pixel size and total dose were 0.5 Å and 60 e− Å−2. Data for the PSI1-IsiA13 samples were collected using a 200 kV Talos Arctica microscope (Thermo Fisher) equipped with a K2 direct detector (Gatan). Data were obtained at a defocus range of −1.2 to −2.5 µm, with 130,000 × magnification and a pixel size of 0.52 Å.

Image processing
Beam-induced motion in the raw movies was corrected using MotionCor 2.151. During PSI3-IsiA43 image processing, the contrast transfer function (CTF) parameters of summed images were estimated using Gctf52. Image processing was further performed using Relion 3.146. Approximately 2000 particles were manually picked from 1800 images and subjected to 2D classification. The best classes were selected and used as references for the reference-based autopicking. After particle picking, 2D classification was performed, and high-quality classes were selected. A total of 310,779 selected particles were used for 3D classification. After 3D classification, 95,950 particles from the class exhibiting a complete IsiA double ring were selected for refinement. The PSI3-IsiA43 complex was finally constructed at a resolution of 3.4 Å.

Image processing of PSI1-IsiA13 followed the same steps as that of PSI3-IsiA43. After excluding the false-positive and false-negative particles through 3D classification, the selected class containing 55,670 particles was constructed at 4.0 Å resolution. The map was subsequently optimized by CTF refinement and Bayesian polishing, resulting in a final resolution of 3.5 Å. The local resolutions of the final maps were calculated using ResMap. The workflows of the cryo-EM analysis of PSI3-IsiA43 and PSI1-IsiA13 are shown in Supplementary Figs. 4, 5, and the data collection and processing statistics are summarized in Supplementary Table 1.

Model building and refinement
The cryo-EM structure of PSI3-IsiA18 from Synechococcus elongatus PCC 7942 (PDB code 6KIG)41 and its single IsiA structure were initially docked onto the PSI3-IsiA43 map using UCSF Chimera for model building. Subsequently, the amino acid sequences were mutated to their counterparts in T. elongatus, and the model was manually adjusted using COOT53 and refined iteratively using Phenix54. The same method was employed for model building of the PSI1-IsiA13 structure. The structural geometries were evaluated using MolProbity55, with statistical details summarized in Supplementary Table 1. Figures were prepared using Chimera56 and PyMOL (Molecular Graphics Systems, LLC).

Multibody refinement and analysis
To explore the flexibility of IsiA subunits in the PSI3-IsiA43 and PSI1-IsiA13 complexes, refined particles from individual datasets were used for 3D multibody refinement in Relion 46. These datasets were generated after CTF refinement and Bayesian polishing procedures. For PSI3-IsiA43 multibody refinement, three rigid bodies corresponding to the PSI core moiety, inner IsiA ring, and outer IsiA ring were assigned by individual soft masks. For PSI1-IsiA13, three rigid bodies were defined as the monomeric PSI core moiety, the inner six IsiAs, and the outer seven IsiAs, using individual soft masks. The initial angular sampling rate, offset range, and offset step were set to 1.5°, six pixels, and 1 pixel, respectively. In the subsequent flexibility analysis using the Relion_flex_analyze program, the motion of the three rigid bodies was decomposed along 18 eigenvectors. The component along the first top eigenvector, comprising ten maps (state001-state010), was output. The translation extents were visualized by superposing the corresponding states.

AFM measurement of thylakoid membranes
Thylakoid membranes were isolated from T. elongatus cells as previously reported 31,57–59 and described below. T. elongatus cells (OD ~0.8) were harvested by centrifugation at 5000 g for 10 min at 4 °C. Cell pellets were washed with a buffer containing 50 mM HEPES-NaOH, pH 7.5, 30 mM CaCl2, and 1% Protease Inhibitor Cocktail, and resuspended in a buffer containing 50 mM HEPES-NaOH, pH 7.5, 30 mM CaCl2, and 800 mM sorbitol. The cells were then broken down using glass beads (150-212 μm) at 4 °C. To obtain pure thylakoid membranes, we separated the membrane fractions in a stepwise sucrose gradient (2.0, 1.5, 1.3, 1.0, 0.5 M) and centrifugation at 40,000 rpm in a SW41 Ti Swinging-Bucket Rotor for 1 h at 4 °C. The Chl-enriched band at the 1.3–1.5 M sucrose interface was collected for AFM imaging.

Thylakoid membranes (2 μL) were adsorbed onto freshly cleaved mica surfaces using 38 μL adsorption buffer (10 mM Tris-HCl, pH 7.5, 150 mM KCl, and 25 mM MgCl2) and were incubated for 1 h at room temperature. After adsorption, the sample was gently rinsed with an imaging buffer (10 mM Tris-HCl, pH 7.5, 150 mM KCl) to remove non-adsorbed thylakoid membranes. High-resolution imaging was performed in liquid at room temperature using dynamic AFM tapping mode (AC mode) on a JPK BioAFM (Bruker, USA) equipped with an ULTRA S scanner and Ultra-Short Cantilever probe (k=0.3 N/m, NanoWorld). The cantilevers were driven at a resonance frequency of 130–140 kHz, and AFM imaging was conducted with a target (setpoint) amplitude of 1.2 V. Scanning was performed at 3 Hz scanning frequency and 512 × 512 pixel resolution. The tip spring constant was routinely calibrated.

AFM images were processed and analyzed using JPK SPM Data Processing software (v 6.1.198). The center-to-center distances between adjacent PSI trimeric protrusions were measured from multiple membrane patches obtained from different samples. These measurements are shown as orange data points in Fig. 5c. For comparison, black data points and a fitted curve were generated based on the previously established model that correlates the center-to-center distances between PSI trimers with the number of surrounding IsiA rings. The distribution of the measured distances is illustrated by a violin plot (gray area shown in Fig. 5c), indicating the frequency of different values 31,44.

FRET analysis
FRET analysis was performed based on the standard FRET theory 7. The rate constant (kFRET) was calculated using the equation kFRET = (C K2) / (n4 R6). Here, C is derived from the spectral overlap integral between the donor and acceptor Chls, K is the dipole orientation factor, n represents the refractive index, and R is the distance between the central magnesium atoms of the two Chls. For energy transfer between Chl a molecules, C was set to 32.26, and n was taken as 1.55, as estimated in ref. 60. The dipole orientation factor (K2) is calculated as K2 = [ûD · ûA − 3(ûD · R DA)(ûA · R DA)]2. Here, ûD and ûA are unit vectors representing the transition dipoles of the donor and acceptor Chls, derived from the coordinates of NB and ND atoms. RDA is the unit vector pointing from the donor to the acceptor Chl’s central magnesium atoms. FRET rates were estimated computationally using Kim’s algorithm implemented in Python (v3.8) and visualized with the interactive platform Gephi61. All calculated K2 and kFRET values for individual chlorophyll pairs are provided in Supplementary files. Fast EET pathways (<20 ps) calculated using different PSI-IsiA structures are listed in Supplementary Tables 2-4.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Si et al. provide valuable insights into the mechanisms by which cyanobacteria adapt to iron-limited conditions through dynamic restructuring of the PSI-IsiA supercomplex. The high-resolution cryo-electron-microscopy structures of PSI -IsiA 3 43 and PSI -IsiA , along with the confirmation of their presence in native membranes via atomic force microscopy and the assessment of energy transfer efficiency through Förster resonance energy transfer (FRET) analysis, collectively demonstrate the critical role of these supercomplexes in optimizing light harvesting and energy transfer. In particular, the formation of a double-layered IsiA ring appears to represent an adaptive strategy for coping with extreme iron deficiency, while the structural flexibility of the outer IsiA layer emerges as a key feature facilitating efficient energy transfer under varying environmental conditions, as highlighted by the authors.\n\nThe findings presented in this manuscript are compelling. However, several ambiguities remain in the description of the mechanism and the results of the analysis that should be addressed before the manuscript can be considered for publication.\n\n1. The observed blue shift of the Q absorption peak is used as an indirect indicator of increased IsiA expression; however, the evidence presented is insufficient to conclusively support this assumption. To more reliably assess IsiA expression levels, quantitative analyses using mass spectrometry or other suitable biochemical methods are necessary.\n\n2. In the Discussion, the authors hypothesize that the formation of the double IsiA ring serves as a mechanism by which cyanobacteria adapt to severely iron-deficient environments. To reinforce this hypothesis, it would be valuable to include experimental data collected under conditions where iron levels are restored following deficiency. Demonstrating structural reversibility upon iron repletion would allow for a more definitive correlation between iron availability and supercomplex architecture.\n\n3. Given the 3.4–3.5 Å resolution, it is difficult to determine inter-chlorophyll distances (e.g., Mg–Mg) and the orientation of diphenyl groups with Å-level precision. Nevertheless, the FRET analysis and the interpretation presented in Figure 6 appear to rely on structural parameters beyond the resolution limit. It would be appropriate for the authors to acknowledge this limitation in the text and clarify the extent to which their conclusions are sensitive to such fine structural details.\n\n4. In FRET analysis, the dipole orientation factor (K2) is calculated based on structural information. The calculated K2 and rate constant (k ) values for all chlorophyll pairs should be provided in the Supplementary Information for transparency and reproducibility.\n\n5. On page 14, the authors state that “the rotational shift of the outer IsiA ring has little effect on the efficiency of outer-to-inner IsiA EET.” However, according to Förster theory, EET efficiency is highly sensitive to the relative orientation of transition dipole moments, which raises doubts about this conclusion. The authors should provide quantitative data or simulations demonstrating that the reported rotational shift truly has minimal impact on EET efficiency.", "reviewer_2": "This study provides a large amount of novel data about the interesting topic of the membrane complexes produced by cyanobacteria under iron starvation or high intensity light exposure. This is a topic of great interest to the photosynthesis community. This seems to be the first high-resolution structure of the double-ring structures that IsiA can form around PSI - which is a novel finding. There is a clear comparison of the newly obtained structures of PSI3-IsiA43 and PSI1-IsiA13, with a good level of detailed descriptions in the text with carefully constructed main-text figures, supported with appropriate supplementary figures. The AFM data and FRET analysis is crucial for extending the scope of the work and provides valuable new insights into the isiA-PSI system, however, these two aspects lack depth and could be improved by simply by providing further detail, as noted below. I think the work is of high quality and wide interest and I would be supportive of publication after the issues listed below have been addressed.\n\nThere are some significant issues that should be addressed -\n\n- The analysis of AFM images including the measurements of centre-to-centre distances between PSI complexes, in Figure 5, is well done. The method is consistent with high-quality measurements from other AFM studies, including the authors' previous work. This is a valuable analysis and gives real insight into the relationship between the cryoEM structures of IsiA-PSI (extracted complexes) and the in-membrane structures of IsiA-PSI. However, the AFM analysis lacks depth and certainty and it could be improved upon.\n\n- It is disappointing that only one field-of-view is shown (one AFM image, Fig 5A and a zoomed in version in Fig 5B). This is a weakness of the paper. Ideally, a gallery of several AFM images from different membrane patches from one biological sample and/or from different samples should be shown in the supplementary information and referred to in the main text. This is important to show that the data presented in the main text is representative of a typical membrane and not just one example. Any supplementary AFM data provided would not need to be of the high resolution and it would be acceptable if the quality was lower so long as the location of the protrusions of PSI trimers can be defined.\n\n- There should be more clarity over how the graph in Figure 5C was generated. It is unclear what each orange datapoint represents (one single distance measurement from AFM data?), or the black datapoints (measurements from the models?) or the error bars of the black datapoints (provide n=? And SD=?), or the grey shaded area of the graph. It is also whether this graph represents measurements from only one AFM image (Figure 5A) or from several AFM images. Currently there is no information about how these measurements were made in either the figure caption or the Methods; there is only a citation of the authors' previous AFM studies, ref 31 and 44. This omission is an oversight and the authors should be given the opportunity to rectify this. I suggest that this information should be briefly stated in the figure caption with any further detail provided in the Methods.\n\n- The FRET analysis of Figure 6C/D is interesting and valuable because it puts the structural data into the energetic context. The analysis may be of high quality, but unfortunately some information is missing and it lacks depth. It is not clear what each grey line between pigment sites represents. The figure caption states \"showing potential high-efficiency EET pathways (lifetime < 40 ps)\" which could mean that ALL pigment couples with a grey line drawn between them have calculated FRET with a time constant (lifetime) calculated as below 40 ps or it could mean that only some of these connected pigments have this lifetime. I suggest that the author explicitly state in the figure caption which FRET couples are displayed in Figure 6C/D.\n\n- Another weakness of the FRET analysis is that this type of display of the FRET pairs is very binary - either some connection or zero connection. It would be better if the variability of the FRET rates between different pigments could be visualized. For example, the lines between pigments could be colour coded based on how high or low their lifetime is. Or the thickness of the bond between pigments could be varied to represent the FRET rate. This would provide more information to readers about how the EET connections within this complex. I assume the authors have this data because the calculations have been performed already so this should not take much more effort to provide further visualizations, and it would benefit readers. This could be another panel in Figure 6 and/or an additional figure in the supplementary information.\n\n- The Discussion section provides an inadequate level of referencing. There should be clear comparison against the state of the art by citation of appropriate literature - currently there only two references in the entire discussion. There should be several in every paragraph - to provide evidence or context to each claim that is made. Further sentences could be added to the discussion of the structural findings to compare to published works on (i) membrane curvature, (ii) protein subunit assembly, (iii) isiA subunit orientation, (iv) iron starvation.\n\n- It is also disappointing that there is no mention in the discussion section of the significance and wider implications of the findings related to the AFM data or the findings related to the FRET analysis. Of course the focus of this study was the cryoEM structures but it would benefit readers if there was a significant discussion about the how these findings provide new insight about the organization of these complexes within native thylakoid membranes and energy transfer processes within. This is a significant oversight and the authors should be able to do this quite easily.\n\nMinor comments -\n\n- The claim about how IsiA assembles around PSI, related to figure 4, is stated as too much of a certainty because EM data shows a static snapshot of the protein complexes and provides no information about the kinetic progression (assembly over time). It is possible that figure 4 shows fragmentation of a larger complex into smaller ones due to the purification process. Or it is possible that the intermediate size complexes are a common features of the membranes as shown by the AFM data. The two sentences below should be reworded to with some qualifies like \"may/ might\" and the other possibilities should be noted/ discussed. \"Our results indicate that the trimeric PSI core initially assembles with a single IsiA ring containing 18 IsiA subunits. Subsequently, additional IsiA proteins bind to the outside of the inner IsiAs in a continuous manner, either by adding in one direction or extending in both directions, until a closed outer ring is formed (Fig. 4).\"\n\n- minor formatting errors in reference list\n\n- \"the Qy absorption band\" - should clarify to \"the Qy absorption band of chlorophyll a\" (line 113 and 128)\n\n- should label the other bands in the figure of the sucrose gradients (Fig S1c) to give context with the other thylakoid proteins.\n\n- should show a magnified version of the Qy peak of purified isiA-PSI complexes (Fig S2b) to show the peak shift because it is not currently clear. For example, a graph of only 650-700 nm over the same width as the current x-axis.\n\n- In the Methods - Should specify how the purified isiA-PSI was concentrated (which brand of spin concentrators? or pelleting + resuspension?) because this can change protein quality.\n\n- The Discussion should cite figures, as appropriate, to direct readers to the associated evidence.", "reviewer_3": "Iron limitation is a common stress factor for cyanobacteria. To cope with this, they produce a protein called IsiA, which helps them harvest light and protect the photosystems. IsiA forms rings around PSI cores. In this study, PSI-IsiA complexes from Thermosynechococcus elongatus were analysed using cryo-EM. The PSI3-IsiA43 shows a trimeric PSI core surrounded by two IsiA rings. The other (PSI1-IsiA13) has a monomeric core with fewer IsiAs. Atomic force microscopy also showed how these complexes are arranged in membranes. The authors claim this results help to understand how cyanobacteria adjust light harvesting and energy transfer when iron is missing.\n\nThe work is technically sound and the resolution is okay to make the claims the authors do. I however completely lack an interpretation of the physiological relevance or any further characterisation that indicates there might be a benefit or function that goes beyond \"it's an curiosity of nature\". As it stands, the study remains a descriptive structural analysis, and apart from the double-ring architecture, the complex does not differ substantially from previously published PSI-IsiA structures. Without further data addressing physiological function or photophysical properties, I believe the manuscript may be more appropriate for a specialized structural biology journal in its current form."}, "author_response": "Response to Reviewer #1:\nSi et al. provide valuable insights into the mechanisms by which cyanobacteria adapt to iron-\nlimited conditions through dynamic restructuring of the PSI-IsiA supercomplex. The high-\nresolution cryo-electron-microscopy structures of PSI -IsiA and PSI -IsiA , along with the\n3 43 1 13\nconfirmation of their presence in native membranes via atomic force microscopy and the\nassessment of energy transfer efficiency through Förster resonance energy transfer (FRET)\nanalysis, collectively demonstrate the critical role of these supercomplexes in optimizing light\nharvesting and energy transfer. In particular, the formation of a double-layered IsiA ring\nappears to represent an adaptive strategy for coping with extreme iron deficiency, while the\nstructural flexibility of the outer IsiA layer emerges as a key feature facilitating efficient energy\ntransfer under varying environmental conditions, as highlighted by the authors.\n\nThe findings presented in this manuscript are compelling. However, several ambiguities remain\nin the description of the mechanism and the results of the analysis that should be addressed\nbefore the manuscript can be considered for publication.\nResponses: We thank the reviewer for the positive comments and valuable suggestions.\n1. The observed blue shift of the Q absorption peak is used as an indirect indicator of increased\ny\nIsiA expression; however, the evidence presented is insufficient to conclusively support this\nassumption. To more reliably assess IsiA expression levels, quantitative analyses using mass\nspectrometry or other suitable biochemical methods are necessary.\n2. In the Discussion, the authors hypothesize that the formation of the double IsiA ring serves\nas a mechanism by which cyanobacteria adapt to severely iron-deficient environments. To\nreinforce this hypothesis, it would be valuable to include experimental data collected under\nconditions where iron levels are restored following deficiency. Demonstrating structural\nreversibility upon iron repletion would allow for a more definitive correlation between iron\navailability and supercomplex architecture.\nResponses: Thank you very much for these excellent suggestions. We agree with the reviewer\n1\nthat the evidence for using Q absorption peak as an indicator of increased IsiA expression is\ny\ninsufficient. Therefore, to roughly quantify IsiA expression levels, we cultured T. elongatus\ncells under different conditions (iron concentrations) and obtained several cell types, namely\nFe+ cell (cultured under iron-sufficient conditions), Fe- cell-1 (cultured under moderate iron-\nlimiting conditions), Fe- cell-2 (cultured under severe iron-limiting conditions) and Fe-/+ cell\n(Fe- cell-2 further cultured under iron-supplemented conditions). We measured the absorption\nspectra of these cells and performed electrophoresis analysis (SDS-PAGE) of the cell extracts.\nThe results are shown in Fig. S1 in the revised manuscript (also see below). We found that the\nIsiA expression levels increased as the iron concentration in the culture medium decreased (Fig.\nS1a), and the Q absorption peak of cells gradually shifted from 683 nm to 678.5 nm and finally\ny\nto 676 nm (Fig. S1b). Moreover, when the iron concentrations were restored after iron\ndeficiency, the Q absorption peak of Fe-/+ cells shifted back to 681.5 nm, and the IsiA protein\ny\nlevels decreased significantly, similar to those of the Fe+ cells. These data collectively support\nour suggestion that the Q absorption peak could be used as an indicator of increased IsiA\ny\nexpression, in good agreement with previous studies (refs 31, 32, 44).\nSupplementary Figure 1. Iron-dependent IsiA accumulation and reversibility of T. elongatus cells. (a)\nSDS–PAGE analysis of the cell extracts of different types of T. elongatus cells treated with various iron\nconditions. The Coomassie-stained band corresponding to IsiA indicated by an arrow was confirmed through\nmass spectrometry. (b) Room-temperature absorption spectra of cells grown under different iron conditions:\nand Fe⁺ (sufficiency). The Q region is shown enlarged.\ny\nFurthermore, we solubilized and further fractioned the thylakoid membranes purified from Fe-\ncell-1, Fe- cell-2 and Fe-/+ cells by sucrose density gradient ultracentrifugation, and then\ncharacterized the major PSI bands using negative-stain electron microscopy. The results are\nshown in Fig. S2 in the revised manuscript (see below). We found that Fe- cell-1 primarily\nformed the PSI -IsiA complex (Fig. S2b), whereas Fe- cell-2 mainly accumulated PSI -IsiA\n3 18 1 13\nand PSI -IsiA complexes(Fig. S2c, d). In contrast, Fe-/+ cells only formed PSI complexes\n3 43 3\n(Fig. S2e), consistent with our electrophoresis analysis results which indicated limited IsiA\nlevels in Fe-/+ cells. These data demonstrate the structural reversibility of PSI and PSI-IsiA\ncomplexes under varying iron concentrations, supporting our proposed correlation between\niron availability and supercomplex architecture. We have included these newly obtained results\nand modified the text in the revised manuscript.\nSupplementary Figure 2. Characterization of the isolated supercomplexes cultured under different\niron concentrations. (a) Sucrose density gradient ultracentrifugation of thylakoid membranes isolated from\nlimitation), and Fe-/+ (iron restored after severe limitation). Major bands are labeled and the PSI bands (bold\nlabels) were collected for negative staining analysis. (b-e) Negative staining images and 2D classification\nresults of PSI complexes obtained from Fe- cell-1 (b), Fe- cell-2 (c, d), and Fe-/+ cell (e).\n3. Given the 3.4–3.5 Å resolution, it is difficult to determine inter-chlorophyll distances (e.g.,\nMg–Mg) and the orientation of diphenyl groups with Å-level precision. Nevertheless, the\nFRET analysis and the interpretation presented in Figure 6 appear to rely on structural\nparameters beyond the resolution limit. It would be appropriate for the authors to acknowledge\nthis limitation in the text and clarify the extent to which their conclusions are sensitive to such\nfine structural details.\nResponses: Thank you for your suggestion. We have modified the text to acknowledge the\nlimitation and clarify that the FRET rates calculated based on our structures may be biased for\ncertain individual EET pathways. Despite that, we believe that the overall EET networks\ncalculated based on our structures are largely reliable. We compared the chlorophyll\narrangement of IsiA proteins from different PSI-IsiA structures, as shown in Fig. S14 in the\nrevised manuscript (see below). Structural superimposition showed that the position and\norientation of all chlorophyll molecules in IsiAs from different structures are similar. We have\nmodified the text as following: According to the Förster theory, FRET rates strongly depend\non the Mg-Mg distances and mutual dipole moment orientations between two adjacent\nchlorophylls. Although the limited resolutions (3.4-3.5 Å) of our structures do not allow a\nprecise determination of these structural parameters, the position and orientation of each\nchlorophyll molecule in IsiAs are similar to those in previously reported PSI-IsiA structures 40-\n43 (Fig. S14). Therefore, we suggest that while the calculated results may be biased regarding\nseveral individual EET pathways, the overall EET networks calculated based on our structures\nare largely reliable.\n(a-b) Side view (a) and Top view (b) of IsiA subunits from PSI-IsiA complexes of different cyanobacterial\nspecies. IsiA subunits in the PSI -IsiA complex from T. elongatus (this study, pink) and the PSI -IsiA\n3 43 3 18\ncomplexes from T. vulcanus (PDB code: 6K33, orange), Synechococcus sp. PCC 7942 (PDB code 6KIG,\ngreen), and Synechocystis sp. PCC 6803 (PDB code 7UMH, blue) were aligned.\n4. In FRET analysis, the dipole orientation factor (K2) is calculated based on structural\ninformation. The calculated K2 and rate constant (k ) values for all chlorophyll pairs should\nFRET\nbe provided in the Supplementary Information for transparency and reproducibility.\nResponses: Thanks for this suggestion. We have provided the complete output files of the\nFRET calculation for both PSI -IsiA and PSI -IsiA , containing K2 and rate constant (k )\n1 13 3 43 FRET\nvalues, as additional supplementary files (named FRET-rate-monomer-output.csv and FRET-\nrate-trimer-output.csv). Moreover, we have summarized the FRET pairs with rapid excitation\ntransfer (lifetime less than 20 ps) from chlorophylls in outer IsiAs to those in inner IsiAs and\nfrom chlorophylls in inner IsiAs to those in the PSI core in both complexes, as shown in Tables\nS2-4 in our revised manuscript.\n5. On page 14, the authors state that “the rotational shift of the outer IsiA ring has little effect\non the efficiency of outer-to-inner IsiA EET.” However, according to Förster theory, EET\nefficiency is highly sensitive to the relative orientation of transition dipole moments, which\n5\nraises doubts about this conclusion. The authors should provide quantitative data or simulations\ndemonstrating that the reported rotational shift truly has minimal impact on EET efficiency.\nResponses: We thank the reviewer for raising this important issue. Following the reviewer’s\nsuggestion, we have calculated the FRET rates of interfacial Chl pairs using the PSI -IsiA\n3 43\nstructure, in which the outer IsiA ring undergoes a large-scale rotation in our multibody analysis,\nand summarized the FRET pairs with rapid excitation transfer (lifetime less than 20 ps) from\nchlorophylls in outer IsiAs to those in inner IsiAs and from chlorophylls in inner IsiAs to those\nin the PSI core (Table S4 in the revised manuscript). The results showed that the primary\ninterfacial Chl pairs involved in the rapid outer-to-inner IsiA EET are similar to those in the\noriginal PSI -IsiA complex, albeit belonging to different IsiA pairs. For example, rapid FRET\n3 43\nrates (lifetime less than 20 ps) from Chl 508 (outer IsiA) to Chl 516 (inner IsiA) calculated\nbased on one PSI -IsiA structure are present in o-3/i-3, o-6/i-5, o-10/i-8, o-13/i-10, o-17/i-13,\n3 43\no-21/i-16, o-24/i-18 IsiA pairs (Table S3 in the revised manuscript), whereas rapid FRET rates\nfor the same Chl pair calculated based on another PSI -IsiA structure occur in o-1/i-1, o-5/i-\n3 43\n4, o-8/i-6, o-12/i-9, o-15/i-11, o-19/i-14, o-22/i-16, o-23/i-17 IsiA pairs (Table S4 in the revised\nmanuscript). These analyses suggest that the rotational shift of the outer IsiA ring did not lead\nto significant changes in the outer-to-inner IsiA EET efficiency, although the underlying\nmechanism remains to be further explored. We have included these data in our revised\nmanuscript. We recognize that the calculations are not based on accurate structures, and\ntherefore, we have toned down the relevant statements.\nThis study provides a large amount of novel data about the interesting topic of the membrane\ncomplexes produced by cyanobacteria under iron starvation or high intensity light exposure.\nThis is a topic of great interest to the photosynthesis community. This seems to be the first\nhigh-resolution structure of the double-ring structures that IsiA can form around PSI - which\nis a novel finding. There is a clear comparison of the newly obtained structures of PSI3-IsiA43\nand PSI1-IsiA13, with a good level of detailed descriptions in the text with carefully\nconstructed main-text figures, supported with appropriate supplementary figures. The AFM\ndata and FRET analysis is crucial for extending the scope of the work and provides valuable\nnew insights into the isiA-PSI system, however, these two aspects lack depth and could be\nimproved by simply by providing further detail, as noted below. I think the work is of high\nquality and wide interest and I would be supportive of publication after the issues listed below\nhave been addressed.\nResponses: We appreciate the extremely positive feedback and thoughtful suggestions to our\nmanuscript.\nThere are some significant issues that should be addressed -\n- The analysis of AFM images including the measurements of centre-to-centre distances\nbetween PSI complexes, in Figure 5, is well done. The method is consistent with high-quality\nmeasurements from other AFM studies, including the authors' previous work. This is a valuable\nanalysis and gives real insight into the relationship between the cryoEM structures of IsiA-PSI\n(extracted complexes) and the in-membrane structures of IsiA-PSI. However, the AFM\nanalysis lacks depth and certainty and it could be improved upon.\n- It is disappointing that only one field-of-view is shown (one AFM image, Fig 5A and a\nzoomed in version in Fig 5B). This is a weakness of the paper. Ideally, a gallery of several\nAFM images from different membrane patches from one biological sample and/or from\ndifferent samples should be shown in the supplementary information and referred to in the\n7\nmain text. This is important to show that the data presented in the main text is representative\nof a typical membrane and not just one example. Any supplementary AFM data provided\nwould not need to be of the high resolution and it would be acceptable if the quality was lower\nso long as the location of the protrusions of PSI trimers can be defined.\nResponse: This is an excellent suggestion. In the revised manuscript, we have included two\nadditional AFM topographs, which were obtained from different membrane patches and\nbiological replicates, as shown in Fig. S11 in our revised manuscript (see below). These AFM\nimages show individual PSI monomeric and trimeric protrusions in thylakoid membranes and\nthe potential arrangements of double-ring and single-ring PSI-IsiA assemblies based on their\nlateral distances, in agreement with the observations depicted in Fig. 5A.\nSupplementary Figure 11. AFM topographs of native thylakoid membranes isolated from the iron-\n8\nstarved T. elongatus cells (Fe- cell-2) revealing the arrangement of PSI-IsiA supercomplexes. (a, b) Two\ndifferent membrane patches. Right panels: PSI-IsiA complexes with double IsiA rings/layers and single IsiA\nrings are annotated by superimposing the PSI -IsiA , PSI -IsiA (PSI -IsiA with the outer IsiA ring\n3 43 3 18 3 43\nremoved), and PSI -IsiA structures on the AFM image.\n1 13\n- There should be more clarity over how the graph in Figure 5C was generated. It is unclear\nwhat each orange datapoint represents (one single distance measurement from AFM data?), or\nthe black datapoints (measurements from the models?) or the error bars of the black datapoints\n(provide n=? And SD=?), or the grey shaded area of the graph. It is also whether this graph\nrepresents measurements from only one AFM image (Figure 5A) or from several AFM images.\nCurrently there is no information about how these measurements were made in either the figure\ncaption or the Methods; there is only a citation of the authors' previous AFM studies, ref 31\nand 44. This omission is an oversight and the authors should be given the opportunity to rectify\nthis. I suggest that this information should be briefly stated in the figure caption with any further\ndetail provided in the Methods.\nResponse: We agree with the reviewer’s comments. In the revised manuscript, we have\nupdated the Methods section and figure legends to clarify the figure and our approach of data\nanalysis. Specifically, the orange data points represent the center-to-center distances between\nadjacent PSI trimeric protrusions measured from multiple AFM topographs (including Fig. 5\nand new Fig. S11) obtained from different membrane patches in three independent samples.\nThe black points and the fitted black line correspond to the predicted distances based on the\npreviously published correlation between IsiA ring numbers and PSI spacing (ref. 31); error\nbars indicate the standard deviation. The grey shaded area represents a violin plot illustrating\nthe overall distribution and density of the measured distances, related to the orange data points.\nWe have also added detailed descriptions of these measurements in the revised Fig. 5 legend\n(see below, the changes are highlighted in yellow) and Methods section (Line 576-584 in the\nrevised manuscript).\n(c) Analysis of the distances between PSI trimers within the PSI-IsiA supercomplexes from\ndifferent membrane patches (orange data points, n=83), which have been demonstrated to be\nclosely correlated with the numbers of IsiA rings between the neighbouring PSI trimers. Black\ndata points (n=51; SD=1.06, 2.32, 0.72, 1.17, 2.47, 2.13 for the number of IsiA rings ranging\nfrom 0 to 6, respectively) and fitted curve based on the previously established PSI-IsiA model31.\nThe grey shaded area reflects the distribution density of orange data points, highlighting that\nmost complexes contain two IsiA rings. A large proportion of PSI-IsiA supercomplexes are\nshown to possess two IsiA rings, as indicated by three, four, or five IsiA surroundings between\ntwo neighboring PSI trimers (indicated by structural models).\n- The FRET analysis of Figure 6C/D is interesting and valuable because it puts the structural\ndata into the energetic context. The analysis may be of high quality, but unfortunately some\ninformation is missing and it lacks depth. It is not clear what each grey line between pigment\nsites represents. The figure caption states \"showing potential high-efficiency EET pathways\n(lifetime < 40 ps)\" which could mean that ALL pigment couples with a grey line drawn between\nthem have calculated FRET with a time constant (lifetime) calculated as below 40 ps or it could\nmean that only some of these connected pigments have this lifetime. I suggest that the author\nexplicitly state in the figure caption which FRET couples are displayed in Figure 6C/D.\n- Another weakness of the FRET analysis is that this type of display of the FRET pairs is very\nbinary - either some connection or zero connection. It would be better if the variability of the\nFRET rates between different pigments could be visualized. For example, the lines between\npigments could be colour coded based on how high or low their lifetime is. Or the thickness of\nthe bond between pigments could be varied to represent the FRET rate. This would provide\nmore information to readers about how the EET connections within this complex. I assume the\nauthors have this data because the calculations have been performed already so this should not\ntake much more effort to provide further visualizations, and it would benefit readers. This could\nbe another panel in Figure 6 and/or an additional figure in the supplementary information.\nResponses: Thank you very much for your valuable suggestions. We have re-produced Figs.\n10\n6c, 6d by changing the thickness of lines between pigments to represent the FRET rates (thicker\nlines indicate faster FRET rates). In addition, we modified the Fig. 6 legend to clarify that only\npigment pairs with calculated lifetimes less than 40 ps are connected by black lines (see below,\nthe changes are highlighted in yellow). In addition, we calculated FRET rates for all pigment\npairs, and the data are provided in additional supplementary files.\nFig. 6 legend\n(c, d) Structure-based FRET calculations showing potential high-efficiency EET pathways in\nPSI -IsiA (c) and PSI -IsiA (d). Chl molecules are shown as spheres, and the FRET\n3 43 1 13\nprocesses between neighboring chlorophylls with lifetime < 40 ps are illustrated as black lines.\nThe FRET processes with longer lifetime ( 40 ps) are omitted for clarity, and the data can be\nfound in supplementary files. Line width indicates the relative FRET rate of each chlorophyll-\nchlorophyll pair (the wider the line, the faster the rate). The EET pathways involving IsiA-i-6\nare highlighted with a red dashed circle.\n- The Discussion section provides an inadequate level of referencing. There should be clear\ncomparison against the state of the art by citation of appropriate literature - currently there only\ntwo references in the entire discussion. There should be several in every paragraph - to provide\nevidence or context to each claim that is made. Further sentences could be added to the\ndiscussion of the structural findings to compare to published works on (i) membrane curvature,\n(ii) protein subunit assembly, (iii) isiA subunit orientation, (iv) iron starvation.\n- It is also disappointing that there is no mention in the discussion section of the significance\nand wider implications of the findings related to the AFM data or the findings related to the\nFRET analysis. Of course the focus of this study was the cryoEM structures but it would benefit\nreaders if there was a significant discussion about the how these findings provide new insight\nabout the organization of these complexes within native thylakoid membranes and energy\ntransfer processes within. This is a significant oversight and the authors should be able to do\nthis quite easily.\nResponses: We thank the reviewer for the constructive comments. Based on these suggestions,\n11\nwe have expanded the Discussion section and cited additional references in the revised\nmanuscript. Specifically, we added further sentences and citations regarding (i) thylakoid\nmembrane curvature and the photosynthetic supercomplex architecture (line 381-394, refs 8,\n45, 47, 48, 49), (ii) the dynamic assembly and structural variability of PSI-IsiA complexes (line\n407-410, refs 32, 38, 41, 42, 43), (iii) the role of IsiA subunit orientation in complex assembly\nand energy transfer (line 413-418, 424-426, 430-447, refs 32, 33, 36, 38, 39, 50), and (iv)\ncyanobacterial adaptation to iron starvation (line 448-458, refs 31, 44). In the revised\nDiscussion section, we compare our findings with recent studies, and discuss the significance\nand wider implications of our AFM and FRET results. In particular, our AFM results link the\nstructural data to the native state of the PSI-IsiA complexes in thylakoid membranes,\ncollectively suggesting the correlation between photosynthetic complex formation and the\nmembrane curvature. Our multibody refinement and FRET rate calculations collectively\ndemonstrate that PSI-IsiA complexes are dynamically assembled while exhibiting robust\nenergy transfer networks, reflecting an adaptive design principle of cyanobacterial light-\nharvesting systems.\nMinor comments -\n- The claim about how IsiA assembles around PSI, related to figure 4, is stated as too much of\na certainty because EM data shows a static snapshot of the protein complexes and provides no\ninformation about the kinetic progression (assembly over time). It is possible that figure 4\nshows fragmentation of a larger complex into smaller ones due to the purification process. Or\nit is possible that the intermediate size complexes are common features of the membranes as\nshown by the AFM data. The two sentences below should be reworded to with some qualifies\nlike \"may/ might\" and the other possibilities should be noted/ discussed. \"Our results indicate\nthat the trimeric PSI core initially assembles with a single IsiA ring containing 18 IsiA subunits.\nSubsequently, additional IsiA proteins bind to the outside of the inner IsiAs in a continuous\nmanner, either by adding in one direction or extending in both directions, until a closed outer\nring is formed (Fig. 4).\"\nmanuscript according to the reviewer’s suggestion as following: Our results suggest that the\ntrimeric PSI core may initially assemble with a single IsiA ring containing 18 IsiA subunits.\nSubsequently, additional IsiA proteins might bind to the outside of the inner IsiAs in a\ncontinuous manner, either by adding in one direction or extending in both directions, until a\nclosed outer ring is formed (Fig. 4). Alternatively, the observed intermediates may reflect a\nnative feature of incomplete PSI-IsiA complexes within the membranes, or could be a result of\ndisassembly of larger complexes into smaller ones during purification.\n- minor formatting errors in reference list\nResponse: Corrected. Thank you.\n- \"the Qy absorption band\" - should clarify to \"the Qy absorption band of chlorophyll a\" (line\n113 and 128)\nResponse: Corrected. Thank you.\n- should label the other bands in the figure of the sucrose gradients (Fig S1c) to give context\nwith the other thylakoid proteins.\nResponses: Following the reviewer’s suggestion, we have labeled all bands in Fig. S1c (now\nFig. S2a in the revised version).\n- should show a magnified version of the Qy peak of purified isiA-PSI complexes (Fig S2b) to\nshow the peak shift because it is not currently clear. For example, a graph of only 650-700 nm\nover the same width as the current x-axis.\nResponses: Thanks for your suggestion. We have provided a magnified version of the Q peak\ny\nshown in Fig. S2b (now Fig. S3b in the revised manuscript), according to the reviewer’s\nsuggestion.\n13\n- In the Methods - Should specify how the purified isiA-PSI was concentrated (which brand of\nspin concentrators? or pelleting + resuspension?) because this can change protein quality.\nResponses: Thanks for your suggestion. The purified PSI-IsiA complexes were concentrated\nusing Amicon Ultra Centrifugal Filters (Millipore) with 100 kDa cut-off, to final concentrations\nof 2-3 mg ml-1 (in Chl). We have provided this information in the Methods section.\n- The Discussion should cite figures, as appropriate, to direct readers to the associated evidence.\nResponses: Thanks for your suggestion. We have cited appropriate figures in the Discussion\nsection.\nIron limitation is a common stress factor for cyanobacteria. To cope with this, they produce a\nprotein called IsiA, which helps them harvest light and protect the photosystems. IsiA forms\nrings around PSI cores. In this study, PSI-IsiA complexes from Thermosynechococcus\nelongatus were analysed using cryo-EM. The PSI3-IsiA43 shows a trimeric PSI core\nsurrounded by two IsiA rings. The other (PSI1-IsiA13) has a monomeric core with fewer IsiAs.\nAtomic force microscopy also showed how these complexes are arranged in membranes. The\nauthors claim this results help to understand how cyanobacteria adjust light harvesting and\nenergy transfer when iron is missing.\nThe work is technically sound and the resolution is okay to make the claims the authors do. I\nhowever completely lack an interpretation of the physiological relevance or any further\ncharacterisation that indicates there might be a benefit or function that goes beyond \"it's an\ncuriosity of nature\". As it stands, the study remains a descriptive structural analysis, and apart\nfrom the double-ring architecture, the complex does not differ substantially from previously\npublished PSI-IsiA structures. Without further data addressing physiological function or\nphotophysical properties, I believe the manuscript may be more appropriate for a specialized\nstructural biology journal in its current form.\nResponses: We thank the reviewer for the highly positive comments on the quality of our\nstructures and the endorsement of our statements. We agree that the PSI core and the inner IsiA\nmoieties of the complex do not differ substantially from previously published PSI-IsiA\nstructures, which we have mentioned in our manuscript (Fig. S5a/S6a in the original/revised\nversion). However, we would like to point out that this is the first high-resolution structures\nreporting the PSI core bound with double-layered IsiA subunits and, to the best of our\nknowledge, the first study that integrates the structural information, AFM data, and FRET rate\ncalculations for PSI-IsiA complexes.\nOur AFM results link the structural data to the native state of the PSI-IsiA complexes in\n15\nthylakoid membranes, collectively suggesting the correlation between PSI-IsiA complex\nformation and the membrane curvature. Our multibody refinement and FRET rate calculations\ncollectively show that PSI-IsiA complexes are dynamically assembled while exhibiting robust\nEET, which might be a key mechanism for cyanobacteria to maintain efficient photosynthesis\nin changing environments. We believe that our findings provide an important basis for\nunderstanding the physiological functions of PSI-IsiA complexes.\nFurthermore, following the reviewers’ suggestions, we characterized T. elongatus cells and\nPSI-IsiA complexes obtained under different iron concentrations using biochemical and\nstructural methods. Our results indicate the correlation between the iron concentration,\nabsorption spectra of cells, and IsiA protein levels. Moreover, our negative staining results\nstrongly suggest that the PSI core and IsiA proteins reversibly assemble and disassemble in\nresponse to changes in iron accessibility, which might be an important adaptive mechanism for\ncyanobacteria to cope with the iron-limited stress.\nIntegrating these newly obtained results, we have expanded the discussion in the revised\nmanuscript, including the assembly and organization of complexes within native thylakoid\nmembranes, the correlation between iron accessibility and structural reversibility of various\nPSI(-IsiA) complexes, the relationship between complex formation and membrane curvature,\nand the structural dynamics of PSI-IsiA complexes and robust energy transfer processes.\nWe believe that our findings provide mechanistic insights into the assembly and structural\nvariations of PSI core-antenna complexes, which will not only advance current understanding\nof photosynthetic complexes but also their reprogramming to design of photosynthetic modules\nfor biofuel/bioenergy applications, which may attract a broad attention from researchers in\nphotosynthetic, synthetic biology, applied biotechnology, protein design coupled with AI\ntechnologies.\nReviewer #1 (Remarks to the Author):\nThe authors' transparency is commendable and substantially strengthens the manuscript.\nHowever, in reviewing the supplementary files, I noted that the units of the calculated\nquantities, particularly the rate constant, are not explicitly specified. For clarity and\nreproducibility, please add the appropriate units to the column headers (e.g., ps-1). Once\nthis minor revision is addressed, I will be pleased to recommend the manuscript for\nacceptance.\nResponse: Thank you for your positive comments and suggestion. The units of lifetime\nand FRET rate are picosecond (ps) and ps-1, respectively, as previously reported (Jan P.\nGötze & Heiko Lokstein, “Excitation Energy Transfer between Higher Excited States\nof Photosynthetic Pigments: 1. Carotenoids Intercept and Remove B Band Excitations”.\nACS Omega 2023. DOI: 10.1021/acsomega.3c05895).\nWe have checked Supplementary Tables 2-4 in our revised manuscript to ensure that\nthe appropriate units have been added to the column headers, and that these units are\nconsistent with those reported in recently published studies involving the FRET rate\ncalculations, which are listed below.\n1) Sheng, X., et al., Nature Plants, 2019. DOI: 10.1038/s41477-019-0543-4.\nExtended Data Figure 9.\n2) Ishii, A., et al., eLife, 2023. DOI: 10.7554/eLife.84488. Table 5.\n3) Shen, L., et al., Science Advance, 2024. DOI: 10.1126/sciadv.adk7140. Table S3.\nReviewer #2 (Remarks to the Author):\nThe authors have comprehensively addressed all of my comments. I recommend\npublication in the current form.\nResponse: Thank you for your support and positive comments.\nReviewer #3 (Remarks to the Author):\nThe authors improved the interpretation of their results and highlighted more clearly\nthat what they observe is of physiological relevance. I have no more objections to\npublish the improved version of this article.\nResponse: Thank you for your support and valuable comments."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors' transparency is commendable and substantially strengthens the manuscript. However, in reviewing the supplementary files, I noted that the units of the calculated quantities, particularly the rate constant, are not explicitly specified. For clarity and reproducibility, please add the appropriate units to the column headers (e.g., ps-1). Once this minor revision is addressed, I will be pleased to recommend the manuscript for acceptance.", "reviewer_2": "The authors have comprehensively addressed all of my comments. I recommend publication in the current form.", "reviewer_3": "The authors improved the interpretation of their results and highlighted more clearly that what they observe is of physiological relevance. I have no more objections to publish the improved version of this article."}, "author_response": "Response to Reviewer #1: Thank you for your positive comments and suggestion. The units of lifetime and FRET rate are picosecond (ps) and ps-1, respectively, as previously reported (Jan P. Götze & Heiko Lokstein, “Excitation Energy Transfer between Higher Excited States of Photosynthetic Pigments: 1. Carotenoids Intercept and Remove B Band Excitations”. ACS Omega 2023. DOI: 10.1021/acsomega.3c05895). We have checked Supplementary Tables 2-4 in our revised manuscript to ensure that the appropriate units have been added to the column headers, and that these units are consistent with those reported in recently published studies involving the FRET rate calculations, which are listed below. 1) Sheng, X., et al., Nature Plants, 2019. DOI: 10.1038/s41477-019-0543-4. Extended Data Figure 9. 2) Ishii, A., et al., eLife, 2023. DOI: 10.7554/eLife.84488. Table 5. 3) Shen, L., et al., Science Advance, 2024. DOI: 10.1126/sciadv.adk7140. Table S3.\n\nResponse to Reviewer #2: Thank you for your support and positive comments.\n\nResponse to Reviewer #3: Thank you for your support and valuable comments."}]

Decision letter