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In situ cryo-ET structure of phycobilisome–photosystem II supercomplex from red alga

  1. Meijing Li
  2. Jianfei Ma
  3. Xueming Li  Is a corresponding author
  4. Sen-Fang Sui  Is a corresponding author
  1. Key Laboratory for Protein Sciences of Ministry of Education, Beijing Advanced Innovation Center for Structural Biology & Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, China
  2. State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology & Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, China
  3. Department of Biology, Southern University of Science and Technology, China
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Cite this article as: eLife 2021;10:e69635 doi: 10.7554/eLife.69635

Abstract

Phycobilisome (PBS) is the main light-harvesting antenna in cyanobacteria and red algae. How PBS transfers the light energy to photosystem II (PSII) remains to be elucidated. Here we report the in situ structure of the PBS–PSII supercomplex from Porphyridium purpureum UTEX 2757 using cryo-electron tomography and subtomogram averaging. Our work reveals the organized network of hemiellipsoidal PBS with PSII on the thylakoid membrane in the native cellular environment. In the PBS–PSII supercomplex, each PBS interacts with six PSII monomers, of which four directly bind to the PBS, and two bind indirectly. Additional three ‘connector’ proteins also contribute to the connections between PBS and PSIIs. Two PsbO subunits from adjacent PSII dimers bind with each other, which may promote stabilization of the PBS–PSII supercomplex. By analyzing the interaction interface between PBS and PSII, we reveal that αLCM and ApcD connect with CP43 of PSII monomer and that αLCM also interacts with CP47' of the neighboring PSII monomer, suggesting the multiple light energy delivery pathways. The in situ structures illustrate the coupling pattern of PBS and PSII and the arrangement of the PBS–PSII supercomplex on the thylakoid, providing the near-native 3D structural information of the various energy transfer from PBS to PSII.

Introduction

Life on Earth depends on photosynthesis for the conversion of solar energy to chemical energy. Red algae living in deep water can efficiently use dim and green light that cannot be absorbed by plants (Tschudy, 1934). This ability is mainly due to two primary photosynthetic components: the light-harvesting antenna and the photochemical reaction centers. The primary light-harvesting antenna is the phycobilisome (PBS), which is located on the stromal surface of the thylakoid membrane and captures light energy (Gantt and Conti, 1965; Gantt and Conti, 1966). The two reaction centers, photosystem II (PSII) and photosystem I (PSI), are located in the thylakoid membrane. PBS mainly transfers the light energy to PSII, leading to water splitting (Biggins and Bruce, 1989; Ley and Butler, 1977a). PBS could also directly couple with PSI (Mullineaux, 1992; Su et al., 1992; Kondo et al., 2007; Ueno et al., 2017).

PBSs are composed of chromophore-bearing phycobiliproteins (PBPs) and linker proteins, which further assemble into central core and peripheral rods (Gantt et al., 1976; Zilinskas and Greenwald, 1986). Recently, the cryo-electron microscope (cryo-EM) structures of PBS from red algae at resolutions of 3.5 Å (Zhang et al., 2017) and 2.82 Å (Ma et al., 2020). The cryo-EM structure of PBS in red alga Porphyridium purpureum (Ma et al., 2020) shows that the PBS consists of a tricylindrical PBS core, 14 rods (Rod a–Rod g, Rod a’–Rod g’), 8 individual extra PE (αβ)6 hexamers (Ha–Hd, Ha’–Hd’), and 24 individual extra PE α or β subunits. The core contains one top cylinder B, composed of two allophycocyanin (APC) (αβ)3 trimers, and two bottom cylinders (A and A’), each of which is assembled by three APC trimers (A1–A3, A2 and A3 form APC hexamer). The rods consist of phycoerythrin (PE) and phycocyanin (PC) hexamers or only PE hexamers. For example, Rod a is composited of one basal PC hexamer and two distal PE hexamers. Thus, the energy absorbed by Rod transfers unidirectionally from the distal PE to the basal PC, and then funnels to APC in the core, and eventually to the two terminal emitters, including chromophores in the core–membrane linker protein (LCM, also called ApcE) (Capuano et al., 1991; Lundell et al., 1981; Tang et al., 2015) and allophycocyanin D (ApcD) (Glazer and Bryant, 1975; Peng et al., 2014).

PSII is a multi-pigment transmembrane protein complex involved in converting light energy into electrochemical potential energy (Adachi et al., 2008). The X-ray crystal structures of PSII from red alga Cyanidium caldarium (Ago et al., 2016), cyanobacterium Thermosynechococcus elongatus (Nakajima et al., 2018), and the cryo-EM structure of PSII from green alga Chlamydomonas reihardtii (Sheng et al., 2019), the marine diatom Chaetoceros gracilis (Pi et al., 2019), and the higher plants Arabidopsis thaliana (van Bezouwen et al., 2017), Spinacia oleracea (Wei et al., 2016), and Pisum sativum (Su et al., 2017) have revealed the structural basis of energy transfer, electron transfer, and photoprotection within the photosystem. The main subunits of PSII include the reaction center D1, D2 protein, and chlorophyll-a binding proteins, CP43 and CP47. Both CP43 and CP47 subunits have been reported to mediate the energy transfer from PBS to the reaction center (Ueno et al., 2017).

The structural mechanism of energy transfer from PBS to PSII has been studied for many years. The organization of PBS and PSII on the thylakoid membrane has been studied using ultrathin section (Gantt and Conti, 1965; Wanner and Kost, 1980), freeze-fracture analysis (Lange et al., 1990), negative staining (Arteni et al., 2008; Chang et al., 2015; Folea et al., 2008; Hellmich et al., 2014), atomic force microscopy (Liu et al., 2008), and cryo-electron tomography (cryo-ET) (Levitan et al., 2019; Rast et al., 2019; Wietrzynski et al., 2020). PBSs in red algae are tightly arranged in ordered rows on the thylakoid in most regions and randomly packed in some areas (Arteni et al., 2008; Lange et al., 1990; Liu et al., 2008). PBS arrangement in the two regions is dynamically regulated, varying with the cell’s age, the supply of nutrients, and available light (Arteni et al., 2008; Liu et al., 2008; Sagert and Schubert, 1995). However, the stoichiometric ratio of PBS/PSII in red algae and cyanobacteria remains controversial, ranging from 1:1 to 1:4 (Arteni et al., 2008; Cunningham et al., 1989; Ohki et al., 1987; Takahashi et al., 2009), which likely reflects different growth conditions. Different approaches showed that PBS connects with PSII through ApcD, αLCM (the α domain of LCM), and ApcF (Zhao et al., 1992). In particular, ApcD and αLCM are well known as terminal emitters for mediating the transfer of energy from PBS to PSII. The negative staining structure of the purified PBS–PSII supercomplex in the Anabaena sp. strain PCC 7120 proposed that αLCM and ApcF of PBS play important roles in mediating PBS interaction with PSII (Chang et al., 2015). The cross-linking structure of the purified PBS–PSII–PSI megacomplex in the cyanobacteria Synechocystis PCC 6803 identified five interlinks associated with αLCM and PSII (Liu et al., 2013). However, the organization and precise connection between PBS and PSII in the cellular environment are not fully understood.

In situ cryo-ET in combination with cryo-focused ion beam (cryo-FIB) milling is currently a powerful approach for directly dissecting macromolecular structures in the unperturbed cellular environment at molecular resolution (Wagner et al., 2020; Watanabe et al., 2020). The obtained structure is complementary to the structure of the isolated complex and reveals elements of the in vivo organization that can never be obtained through structures of the isolated complex, no matter how high the resolution. Here, by using this approach, we identified the native PBS–PSII supercomplex and the double PBS–PSII supercomplex containing two adjacent PBS–PSII supercomplexes at resolutions of 14.3 Å and 15.6 Å, respectively. These results illustrate the coupling pattern of PBS and PSII and the arrangement of PBS–PSII supercomplexes on the ordered distribution region of the thylakoid membrane, which cannot be determined with other methods. The newly found structural information provides a better understanding of energy transfer from PBS to PSII.

Results

Visualization of PBS and PSII on the thylakoid membrane

The red alga, P. purpureum, was cultured under low-light conditions to mimic its natural habitat (Cunningham et al., 1989) and to increase the population of regularly distributed PBS–PSII supercomplexes (Liu et al., 2008). Cells were harvested in the exponential phase of growth and were immediately vitrified on cryo-EM grids to minimize environmental affection (Kaňa et al., 2014). Cryo-FIB milling was then used to prepare thin lamellae with ~150 nm thickness for cryo-ET data collection. We first imaged the whole cells. The tomogram slice shows that most PBS–PSII supercomplexes are packed in an orderly fashion on the thylakoid membranes (Figure 1—figure supplement 1A). Subsequently, 51 cryo-ET tilt series were collected at higher magnification at the ordered distribution regions for three-dimensional (3D) reconstruction and further sub-tomogram averaging.

Consistent with the conventional transmission electron microscope ultrastructures of other red algae (Gantt and Conti, 1965; Gantt et al., 1968; Tsekos et al., 1996; Wanner and Kost, 1980), PBSs are sandwiched between parallel thylakoid membranes. In the cross-section view, adjacent PBSs were attached to the upper and lower thylakoid membrane in opposite directions (Figure 1A,B,D). In the two orthogonal views, opposite PBSs are stacked into linear rows respectively (Figure 1C,E, Figure 1—figure supplement 1B,C, Video 1, Video 2, Video 3). These PBS rows are arranged in parallel, forming a large two-dimensional pattern covering the thylakoid membrane. In the cross-section view, densities are evident at the bottom of PBSs, which are embedded within the thylakoid membrane. Protrusions are visible on the luminal side (Figure 1A). The features of these densities are consistent with the location and morphology of PSIIs (Ago et al., 2016; Chang et al., 2015; Lange et al., 1990). From this, we can infer that these represent PSIIs. Due to the small size of the extrinsic domain of PSI, we could not distinguish convincing PSI densities in our tomograms.

Figure 1 with 1 supplement see all
Organization of PBS–PSII supercomplexes on the thylakoid membrane.

(A) Representative tomogram slice in cross-section view. The box represents magnified details of PBS, a PSII dimer, the thylakoid membrane, and the lumen. (B) Spatial mapping of the PBS–PSII supercomplex (purple) and 3D segmentation of the thylakoid membrane (blue). (C–E) The magnified three presents perpendicular views of the organization of PBS–PSII in the thylakoid membrane boxed in (B). Thylakoid membrane, light blue; PBS, gray; Lateral hexamer, green; A1–A2 pair, purple; B1–B2 pair, cyan; C1–C2 pair, yellow; connector 1, coral; connector 2, khaki; connector 3, red.

Video 1
Sequential slices back and forth through the representative tomogram in cross-section view.

Related to Figure 1. Scale bar, 100 nm.

Video 2
Sequential slices back and forth through the representative tomogram slice in cross-section view.

Related to Figure 1—figure supplement 1B. The color boxes indicate the two types of the tight perforations. Scale bar, 100 nm.

Video 3
Sequential slices back and forth through the representative tomogram slice in cross-section view.

Related to Figure 1—figure supplement 1C. Scale bar, 100 nm.

To verify the PSII densities, we performed sub-tomogram averaging of two adjacent PBS particles on the same linear array (indicated in the red boxes of Figure 1—figure supplement 1B,C) and generated a structure at a resolution of 15.6 Å (Figure 2A, Figure 2—figure supplement 1A,B). The recently determined 2.82 Å single-particle PBS (PDB code 6 KGX) (Ma et al., 2020) was well-fitted into our sub-tomogram map (Supplementary file 1), except for the individual extra PE β subunit between the Rod e and Rod d' (Figure 2—figure supplement 2A,B). The map’s slice view shows a similar PSII dimer array structure resembling that of the red algae Porphyridium cruentum (Lange et al., 1990; Figure 2—figure supplement 3A,B).

Figure 2 with 5 supplements see all
Overview of the PBS–PSII and double PBS–PSII structures.

(A) The density map of double PBS–PSII structures at a resolution of 15.6 Å, presented in two perpendicular views. The center-to-center distance of two adjacent PBSs is approximately 345 Å. Two random circles indicated by black or red dashed lines mark the six PSII monomers binding with each of the two PBSs. PBS1 is associated with PSII monomer A1, A2. B1, B2, C1, and C'2. PBS2 connects with A'1, A'2, B'1, B'2, C'2, and C’’1. The surface threshold is 0.09. (B) The density map of the PBS–PSII supercomplex at a resolution of 14.3 Å fitted with the single-particle model of PBS (EMDB code EMD-9976, PDB code 6 KGX) and X-ray structure of PSII (PDB code 4YUU), presented in two perpendicular views. The lateral hexamer was fitted with the single-particle model of the Rod a distal PE hexamer. The surface threshold is 0.059. (C) The magnified image shows that the two PsbO subunits bind with each other at the interface of the adjacent PSII dimers (Inset). A map of PSII dimers A and B, segmented from (B) with the same surface threshold level. The arrow indicates the binding site.

To find the most likely PSII atomic resolution structure to fit the subtomogram map, we analyzed all of the reported X-ray models in a phylogenetic analysis (Figure 2—figure supplement 4B). PSII from the red algae C. caldarium (PDB code 4YUU) (Ago et al., 2016) is, evolutionarily, most similar to P. purpureum, and almost all the subunits show high homogeneity (ranging from 35% to 100%) with that of P. purpureum (Figure 2—figure supplement 4A,C). We then docked the X-ray model of PSII from C. caldarium into the sub-tomogram map, which shows a high cross-correlation coefficient (all above 80%) between the model and sub-tomogram map (Supplementary file 1). Thus, we further confirmed these densities as PSIIs.

The overall structure of the PBS–PSII supercomplex

Our sub-tomogram map also reveals a different stoichiometry of PBS and PSII from previous reports (Arteni et al., 2008; Cunningham et al., 1989; Ohki et al., 1987; Takahashi et al., 2009). In the sub-tomogram map, two PBSs (labeled as PBS1 and PBS2) interact with a linear array of PSII dimers (labeled as A, B, C, A', B', C', C", Figure 2A (left panel), Figure 2—figure supplement 3B). Considering the map contains two PBSs, we named the structure double PBS–PSII supercomplex. After mapping this sub-tomogram map back into the tomogram, we observed long linear arrays of the PBS–PSII supercomplexes on the thylakoid membrane (Figure 1B–E). In this array, the PBS/PSII ratio is 1:6, that is, one PBS with six associated monomeric PSIIs periodically repeated along with the linear array. In the double PBS–PSII map, each PBS is directly anchored to a pair of PSIIs dimers, i.e., PBS1 on the A–B pair and PBS2 on the A’–B’ pair (Figure 2A, right panel). The C' dimer connects the A–B pair to the A’–B’ pair, while the two PSIIs in the C' dimer respectively interact with the lateral disc-like densities on both sides of PBS, which have features of PBS hexamer (discussed later), and thus indirectly attach to PBS1 and PBS2 (Figure 2A). The slice view shown in Figure 2—figure supplement 3B indicates that the PSII dimers are arranged repeatedly in parallel. Thus, C and C’’ should also be the PSII dimer, although their densities are weak. The structure shows PBS1 is associated with PSII monomer A1, A2, B1, B2, C1, and C’2, and PBS2 connect with A’1, A’2, B’1, B’2, C’1, C’’2. Therefore, each PBS is associated with six monomeric PSIIs.

To study the geometry of the PBS–PSII supercomplex, we manually picked 34,380 individual PBSs with the interacting PSIIs particles. These were aligned and averaged into a structure at a higher resolution, 14.3 Å (Figure 2B, Figure 2—figure supplement 1A,C). In the PBS–PSII map, the PSII dimers (PSII dimers A and B) are clearly shown. With a large threshold, the other two PSIIs corresponding to PSII dimer C in double PBS–PSII are visualized in the map and tomogram slice view (indicated with red stars in Figure 2—figure supplement 3C,D). The distance between the PSII dimers A and B is approximately 12.4 nm, and the face planes of the two PSII dimers make a lateral angle of approximately 14° with the plane of the PBS central core. The centers of the two PSII double dimers shift approximately 3 nm along the plane of the PBS central core, which makes the twofold symmetry axes of PBS and 6 PSIIs coincide (Figure 2—figure supplement 5).

The improved PBS–PSII structure enables us to confirm that PSII dimers A and B bind with each other through a rigid density. By analyzing the fitting results, we suppose the density is most likely contributed by the two adjacent PsbO subunits: one is from the PSII monomer 1 of the PSII dimer A and the other from the PSII monomer 4 of the PSII dimer B (Figure 2, Figure 2—figure supplement 2C–E). However, we are limited by the current resolution and could not exclude that other factors may contribute to the density. The PsbO protein is found in PSII of all oxygenic organisms ranging from cyanobacteria and red algae to higher plants and serves as the manganese-stabilizing protein directly involved in the stability of photosynthetic water-oxidizing complex (Ifuku and Noguchi, 2016; Popelkova and Yocum, 2011). Our results suggest that PsbO could play an additional role in stabilizing adjacent PSII dimers in PSII dimer rows.

To analyze the lateral densities on both sides of PBS connecting PBS to PSII dimer C, we further improved the resolution of the PBS–PSII supercomplex to 13.2 Å after post-processing from the 14.3 Å resolution map (Figure 2—figure supplement 1D). The slice view of the PBS–PSII map shows that the densities are round discs with solid densities in the center (Figure 2—figure supplement 3E–G). These features are reminiscent of the structure of the extra hexamer found in the single-particle cryo-EM structure of P. purpureum PBS. Moreover, the lateral densities are of the same size and shape as the extra hexamer. As all eight extra hexamers in P. purpureum PBS are PE hexamers, we hypothesize that the lateral density could be contributed by a PE hexamer, which is referred to as the lateral hexamer (Figure 2B). Further analysis indicated that the lateral hexamer connects with the bottom hexamer of Rod a and the second hexamer of Rod e, which are the PC and PE hexamers, respectively (Figure 2—figure supplement 3E).

Interaction pattern between PBS and PSII

In the PBS–PSII map, extensive interfaces between PBS core and PSII are observed. To better analyze the potential interactions, we docked the atomic models of PBS and PSII into the map and extracted the densities of PBS core cylinders A, A' and PSII dimers A, B (Figure 3A,B; Figure 3—figure supplement 1). ApcD and αLCM are two well-documented terminal emitter subunits of PBS (Zhao et al., 1992; Zlenko et al., 2019). We observed the interfaces between αLCM, ApcD, and PSII dimers A, B from the surface clip views (Figure 3, Video 4).

Figure 3 with 1 supplement see all
The connections between PBS and PSIIs.

(A, B) A segmentation map of two basal cylinders of the PBS core (labeled as a and a', respectively) and PSII dimers A and B (each monomer is labeled as A1, A2, B1, and B2, respectively). Each basal cylinder consists of three APC trimers, a1, a2, and a3. This map was segmented from the PBS–PSII supercomplex at a surface threshold level of 0.065. (C–F) Surface clip views of the segmented PBS–PSII supercomplex show the details of the interaction between the αLCM, ApcD, and CP43, CP47. The clip planes were indicated in (A). The surface threshold was 0.065. CP43A1, CP43 of PSII dimer A1; CP47B1, CP47 of PSII dimer B1.

Video 4
Clip views of the segmentation map of two basal cylinders of the PBS.

Related to Figure 3.

As was shown in the proximal and middle clip views, there is a wide interface between αLCM and CP43 of PSII dimer A1 (CP43A1), as well as a relatively small interface between αLCM and CP47 of PSII dimer B1 (CP47B1) (Figure 3C–E). These observations suggested the interactions between αLCM with CP43 and CP47, consistent with the energy transfer from PBS to CP43 and CP47 detected in red algae and cyanobacteria (Ueno et al., 2017). The different sizes of the interface may provide a structural view that energy transfer from PBS to CP43 is the main pathway in red alga (Ueno et al., 2017). The other terminal emitter subunit ApcD was also involved in energy transfer from PBS to PSII (Ashby and Mullineaux, 1999; Calzadilla et al., 2019; Ley and Butler, 1977b). The middle and distal clip views showed an interaction interface between ApcD and CP43A1 (Figure 3E,F), which may mediate the energy transfer from ApcD to PSII.

Supplementary interactions intermediated by three connector proteins

After docking the atomic models of PBS, PSII dimer, and two lateral hexamers into the density map of PBS–PSII supercomplex, we still observed three extra densities that are not occupied by any model. Two of them are associated with the PBS and PSII (Figure 4—figure supplement 1A), and the third one is associated with the lateral hexamer and PSII (Figure 4—figure supplement 1B). Since we could not identify the proteins corresponding to these densities, we temporarily deemed them ‘connectors’ (connectors 1–3; Figure 4, Figure 4—figure supplement 2A–E). Nevertheless, their positions in the map suggest that these proteins likely participate in the formation of the PBS–PSII supercomplex (Figure 4) as well as the assembling of the PBS–PSII array on the thylakoid membrane (Figure 1C–E).

Figure 4 with 2 supplements see all
Three connector proteins in the PBS–PSII supercomplex.

(A) Overall structure of the PBS–PSII supercomplex (surface threshold of 0.065), highlighting the PBS core, Rod a of PBS, connector 1, and connector 2 between PBS and PSII. (B, C) The magnified clip views of connector 1 show that connector 1 interacts with the β2 subunit of the a3 trimer layer and the CP47, D1, and D2 subunits of PSII monomer B2 (labeled with CP47B2, D1B2, and D2B2, respectively). (D, E) The magnified clip views of connector 2 show that connector 2 mediates the connections between the bottom hexamer of Rod a and D1, D2, and CP47 of PSII monomer B1 (labeled with D1B1, D2B1, and CP47B1, respectively). (F) The overall structure of double PBS–PSII supercomplex (surface threshold = 0.09) highlights the linkage of connector 3 between PBS’s lateral hexamer and PSII’s bridging dimer C shown in Figure 2A. (G, H) The density of connector 3 and its putative connections with PBS’s lateral hexamer and PSII from two perpendicular views. According to the slice view of the PBS–PSII supercomplex, some densities of connector 3 are inserted into the central cavity of the lateral hexamer (Figure 4—figure supplement 2E); this cavity density is not colored here.

We segmented out the three connectors from the PBS–PSII supercomplex map and find that they are all rod-like structures, but different in size (Figure 4—figure supplement 2). The surface clip views of connector 1 show that connector 1 contacts the β2 subunit of the PBS core layer a3 and the CP47 of PSII monomer B2 (CP47B2), as well as D1B2 and D2B2 (Figure 4B,C). Connector 2 is smaller than connector 1 (Figure 4D,E, Figure 4—figure supplement 2A–C). One side of connector 2 interacts with the basal hexamer of Rod a, and the other side with D1B1, D2B1, and CP47B1 in the neighboring PSII monomer B1 (Figure 4D,E). Thus, both connector 1 and connector 2 connect with PBS and PSII, suggesting that they may consolidate the anchor of PBS on PSII dimer.

Different from connectors 1 and 2, connector 3 mediates a connection between the lateral hexamer and the bridging PSII dimer C. The density map of connector 3 shows that connector 3 could be divided into one small part and one big part (Figure 4—figure supplement 2D,E). The small part is buried in the lateral hexamer’s central cavity, and the big part extends out to connect with PSII. This structural feature is very similar with the structure of some rod linker proteins, such as LR6 of P. purpureum PBS, with the N-terminal rigid domain occupying the central cavity of a hexamer and the C-terminal region extending out, suggesting that connector 3 might be the linker protein of the lateral hexamer. The structure indicates that the lateral hexamer together with connector 3 stabilizes PBS’s connection with PSII in the interconnected array of the PBS–PSII supercomplex.

Discussion

Organization of PBS and PSIIs

PBS is classified into different structural types, including hemidiscoidal (Chang et al., 2015), hemiellipsoidal (Arteni et al., 2008), block type (Zhang et al., 2017), bundle type (Guglielmi et al., 1981) and rod type (Chen et al., 2009). These results in diverse organizational structures between PBS and PSII. Hemidiscoidal PBSs of the Synechocystis strain PCC 6803 and the Anabana sp. strain PCC 7120 are associated with one PSII dimer (Chang et al., 2015; Rast et al., 2019). The rod-like AmPBS is composed of four hexamers and is adjacent to one PSII dimer (Chen et al., 2009; Niedzwiedzki et al., 2019). The hemiellipsoidal PBS connects with monomeric PSII at a ratio from 1:1 to 1:4 (Arteni et al., 2008; Cunningham et al., 1989; Lange et al., 1990; Ohki et al., 1987; Takahashi et al., 2009). Using in situ cryo-ET, our work reports the native PBS–PSII supercomplex structure of red algae. The structure reveals a different ratio (1:6) of hemiellipsoidal PBS and PSII on the native thylakoid membrane (Figure 5A,B). Each PBS directly interacts with two PSII dimers and indirectly interacts with two PSII monomers from two peripheral bridging PSII dimers, forming the interconnected array of the PBS–PSII supercomplex with the support of three unassigned connectors. With 14° rotation and 30 Å shift of PSII dimer pair relative to PBS core, the terminal emitters, ApcD and αLCM, in the two symmetric a2a3 and a2'a3' hexamers of the core, connect with CP43 of PSII monomers A1 and B2, and CP47 in PSII monomers B1 and A2 (Figure 5). A similar geometry of the PBS–PSII supercomplex was reported in cyanobacteria and red algae possessing hemidiscoidal PBS, suggesting that hemidiscoidal PBSs use the same strategy for energy transfer from PBS to PSII (Arteni et al., 2009; Arteni et al., 2008). This structure also enables the extra lateral hexamers in P. purpureum to associate with PSII monomers C1 and C'2 through connector 3 (Figure 5A,B).

Schematic model of the PBS–PSII supercomplex.

(A) The front view of the schematic model of the PBS–PSII supercomplex. For PBS, only core and Rod a are represented for clarity. In the PBS core, a and a' represent the two basal cylinders. Each cylinder consists of three APC trimers. b represents the top cylinder containing two APC trimers. (B) The side view of (A). Each PBS interacts with six PSII monomers: the two basal cylinders a and a' of the PBS core directly connect with PSII monomers A1, A2, B1, and B2. The connections are consolidated by connector 1 and connector 2; the lateral hexamers indirectly interact with the PSII monomers C1 and C'2 in the two peripheral PSII bridging dimers C and C' through connector 3. Two PsbO subunits from the adjacent PSII dimers bind with each other and may promote stabilization of the PBS–PSII supercomplex. The PBS–PSII supercomplexes regularly stack face-to-face into the interconnected arrays, forming the organized network on the thylakoid membrane. (C, D) The detailed connections between PBS and PSII. αLCM interacts with CP43 and CP47 of PSII monomer, which may provide a way to funnel light energy to the reaction center. ApcD connects with CP43, suggesting another energy transfer way. (D) Highlights the connections of αLCM - CP43A1, αLCM–CP47B1, and ApcD–CP43A1. CP43A1, CP43 of PSII dimer A1; CP47B1, CP47 of PSII dimer B1.

In this work, we focused on the PBS–PSII structures in the ordered distribution regions, where both PBS and PSII are neatly arranged according to a strict stoichiometry of 1:6. Previous research conducting fraction freezing analysis (Lange et al., 1990) has found that the PBS–PSII complexes on random distribution regions could have lower stoichiometry (between 1:1 and 1:4), which are represented as an PSII monomer, dimer, trimer, or double dimer. These observations show the possible diversity of PBS–PSII organization on the thylakoids.

The connection between PBS and PSIIs

Extensive studies have shown that αLCM and ApcD are the terminal emitters of the energy coupling between PBS and PSII (Zhao et al., 1992; Zlenko et al., 2019). In our structures, we detailed the connections of αLCM and ApcD with PSII, which contribute to the energy flows from PBS to PSII (Figure 3). ApcF was also reported to be involved in the energy transfer in cyanobacteria (Calzadilla et al., 2019; Chang et al., 2015). However, we could not find a solid connection between ApcF and PSII. Thus, it is very likely that ApcF only plays an auxiliary role for direct energy transfer to reaction centers in red algae.

Additionally, these in situ structures reveal that three extra connectors are associated with PSII by the PBS core, Rod a, and the lateral hexamer, respectively. While the current resolution limited additional detailed analysis of the connectors, we attempted to estimate the molecular weights of these connectors at around 30–80 kDa according to the size and shape (Figure 4—figure supplement 2).

We found a class of PSII dimer C (PSII dimer C, C', and C" in Figure 2A) that does not directly bind to PBS but is closer to the lateral hexamer. Since PSII dimer C can only absorb energy from the independent lateral hexamer instead of the whole PBS through connector 3, they could have fewer bilins feeding into them than the other PSIIs and could become saturated with light at higher intensities. If the PSII dimer C is more sensitive to light intensity than the others, it could be involved in the transition between the ordered distribution region and the random distribution region. The transient absorption spectroscopic measurements and time-resolved fluorescence spectroscopy at low temperatures (77k or 4k) could help confirm this hypothesis.

The energy transfer from PBS to PSII could follow multiple pathways (Figure 5). The two terminal emitters, αLCM and ApcD, funnel light energy to PSII by distinct structural connections. The chromophore of αLCM mediates energy transfer via interaction with CP43 and CP47. In contrast, the chromophore of ApcD is mediated by interactions with CP43. Since connector 2 connects with both the basal PC hexamer of Rod a and CP47 of PSII, we speculate that alternative routes of energy transfer bypassing the PBS core to PSII could exist: connector 2 and connector 3 could mediate the energy flow from the basal hexamer of Rod a to PSII and from lateral hexamer to PSII, respectively. Ueno et al. have reported light energy flows from CP43 to CP47 by energy transfer between the neighboring PSII monomers (Ueno et al., 2017). Our in situ structure reveals that αLCM transfers energy to CP43 as well as the neighboring CP47, may suggesting that αLCM provides the modulation activity of energy flow between CP43 and the neighboring CP47. However, it is currently unclear whether the connector proteins play a specific role in the energy transfer between PBS and PSII, and this topic requires further study.

The perforations and stairs of the thylakoids

In several tomograms, we observed the thylakoid membrane branching and fusion regions (Figure 6, Video 5). Within the limited thickness of the tomogram, we observed a small area of thylakoid membranes present branching and fusion events (Figure 6A). The two parallel thylakoid membranes are split into two branches. One branch approaches and fuses with another branch from the neighboring thylakoid membrane (Figure 6A,B). The 3D segmentation model shows that the branching and fusing membranes built ‘stairs’ between two neighboring thylakoid membranes (Figure 6C,D). On the top of the ‘stairs’ is the large perforation on the thylakoids (Figure 6E). These structures are very similar to the thylakoid membrane architecture in the cyanobacteria Prochlorococcus (Ting et al., 2007), Synechococcus sp. PCC 7942 and Microcoleus sp. reported by Nevo et al., 2007. They also observed perforations as well as branching and fusion of the membranes, which resulted in a highly connected network to allow water-soluble and lipid-soluble molecules to diffuse through the entire membrane network (Nevo et al., 2007). Thus, it is reasonable to deduce that the perforations and stairs of the thylakoid membrane in the red alga might play the same role, although it needs to be further investigated.

The thylakoid membrane builds perforations and stairs by branching and fusing with the neighboring membrane.

(A) Typical tomogram slice. The white box indicates the tomogram region shown in Figure 1A; red box represents the membrane branching and fusing area. Scale bar, 100 nm. (B) Sequential slices back and forth through the representative tomogram in the red box area of (A), showing the membrane branches off one membrane and fuse with the neighboring membrane. The black arrow shows the fusion membrane; Red arrow shows the branching event. Scale bars, 50 nm. (C) 3D segmentation model of the red box area of (A) in the absence of the PBS–PSII supercomplexes to show the 3D morphology of the thylakoid. The two-line arrow indicates the membrane stairs. The numbers label different layers of membrane. (D) The clip view of the 3D segmentation model. The clip position is showed in the right panel of (C). The arrows show the membrane stairs. (E) The membrane top view shows the perforation on the top of the membrane stair. Figure accompanied by Video 5.

Video 5
Sequential slices back and forth through the representative tomogram slice to show thylakoid membrane branching and fusing areas.

Related to Figure 6A–E. Scale bar, 20 nm.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Porphyridium purpureum)Porphyridium purpureum UTEX 2757https://utex.org/products/utex-lb-2757UTEX Culture Collection of Algae 2,757
Gene (Porphyridium purpureum)uniprothttps://www.uniprot.org/uniprot/UP000324585Proteome
Software, algorithmSerialEMhttps://bio3d.colorado.edu/SerialEM/Version 3.1.8Data collection
Software, algorithmChimerahttps://www.cgl.ucsf.edu/chimera/Version 10.15Visualization
Software, algorithmRelionhttps://www3.mrc-lmb.cam.ac.uk/relion/Version 2.1Subtomogram averaging
Software, algorithmIMODhttps://bio3d.colorado.edu/imod/index.htmlVersion 4.9Tomogram reconstruction
Software, algorithmTOMO3Dhttps://sites.google.com/site/3dem/imageprocessing/tomo3d
Version 2.0Tomogram reconstruction
Software, algorithmI3https://www.electrontomography.org/Version 0.9.3Subtomogram averaging

Cell culture and cryo-EM sample preparation

Request a detailed protocol

Porphyridium purpureum (from UTEX Culture Collection of Algae, UTEX 2757) cell was cultured in Bold 1NV:Erdshreiber (1:1) half-seawater medium, bubbled with sterilizing filtered air at 22°C, under a light–dark periodic ratio of 16 hr:8 hr, with a white light flux of about 37 μmol photons•m−2•s−1. Then red algal cells were harvested at about 1.5–3.0 months by centrifuge at 6000 g at room temperature for 10 min under dark, and the pellet was washed and re-suspended with fresh half-seawater culture medium under dark.

We used holy-carbon copper grids (Quantifoil R1.2/1.3, 200 mesh) for the cryo-EM sample preparation. Cryo-EM grids were prepared with Leica EM GP (Leica Company) at 20°C and 100% humidity. To minimize the environmental affection (Kaňa et al., 2014), we first prepared the plunge freezing equipment. Then red algal cells are harvested and immediately vitrified in the Leica EM GP chamber without light. A drop of 4 μL culture medium was added to the glow-discharged grids. After being blotted, the grids were plunged into liquid ethane and stored in liquid nitrogen. We prepared four to six grids for each session. Each grid took less than 1 min. Moreover, we covered a layer of foil paper on the cell volume to protect them from the light.

Cryo-FIB lamella preparation

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With the modified workflow, as Schaffer et al., 2017 reported, cryo-EM grid was first transferred to Helios NanoLab G3 (FEI) system. A layer of Au was sputtered to the surface of cryo-EM sample to increase the conductivity. A layer of protective organometallic platinum was then deposited on the top of the sample with the GIS system. The working distance was 10 mm, and the GIS temperature was set to 46°C. Ga2+ ion beam was used to milling the cells at a 5° stage tilt. The beam current for rough milling was 0.79 nA and gradually decreased to 40 pA. The lamella was finally polished to about 150 nm in thickness with the beam current of 24 pA.

Cryo-ET data collection

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The cryo-ET data were collected with Titan Kiros Microscopy (Thermo Fischer Scientific) operated at a voltage of 300 kV and equipped with a Cs corrector, GIF quantum energy filter, and K2 Summit direct electron detector (Gatan Inc). All tilt series were recorded from 60° to –60° with SerialEM software (Mastronarde, 2005). The recording state was at a nominal magnification of 33,000× in counting mode with a pixel size of 3.421 Å•pixel–1. Each stack was exposed for 2.4 s with an exposure time of 0.3 s per frame and recorded as a movie of 8 frames, resulting in the total dose rate of approximately 1.927 electrons per Å2 for each stack. The tomographic tilt series were recorded from −50° to +66° with an increment of 2° using the unidirectional strategy. On average, 6 frames were collected for each image resulting in a total dose between 100 e2 and 110 e2 per tilt series. GIF was set to a slit width of 20 eV. The defocus ranged from –2.8 μm to –5.5 μm. MotionCor2 program was used to correct the beam-induced motion (Zheng et al., 2017).

Cryo-ET reconstruction and sub-tomograms averaging

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Fifty-one tilt series were aligned with the patch-tracking method in IMOD software (Kremer et al., 1996). The tomograms were reconstructed with TOMO3D scripts (Agulleiro and Fernandez, 2015). The tomograms reconstructed with simultaneous iterative reconstruction technique were used to manually particle-picking. To get high-resolution structures, we performed sub-tomogram averaging. Five thousand and thirty-seven sub-tomograms were firstly picked and aligned with i3 software to generate an initial model (Winkler et al., 2009). Tomograms reconstructed with weighted-back projection were used for further sub-tomogram averaging analysis. Then 75,310 manually picked sub-tomograms were extracted with RELION software (Bharat et al., 2015). CTF correction was estimated with CTFFIND4 (Rohou and Grigorieff, 2015) implemented in RELION. Dose damage compensation was performed with the script provided in the RELION tutorial. The first round of auto-refinement was performed with the binning four sub-tomograms. For the reconstruction of the double PBS–PSII supercomplex, the coordinates of first-round auto-refinement were shifted and carried on another round of 3D classification with global search. The good classes were selected and performed auto-refinement with the binning two sub-tomograms. After refinement, a resolution map at 15.6 Å was achieved. To improve the resolution, we reconstructed the PBS–PSII supercomplex with the refined coordinates and Euler angles obtained in the first round of rough refinement. These sub-tomograms were performed 3D classification without alignment. The good classes were chosen to do another round of auto-refinement with local search at 1.8 degrees. Finally, the sub-tomograms of the PBS–PSII supercomplex without binning were refined to 14.3 Å and postprocessed to 13.2 Å. During the auto-refinement, the datasets were automatically divided into two halves by RELION. The resolution was estimated using the gold-standard Fourier shell correlation with 0.143 criteria by ResMap (Scheres and Chen, 2012).

Atomic model fitting and analysis

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To analyze the double PBS–PSII and PBS–PSII sub-tomogram averaging maps, the atomic model of PBS built with single particle analysis and crystal model of PSII (PDB code 4YUU) were docked into the sub-tomogram averaging maps with ‘fit in map’ command in UCSF Chimera (Pettersen et al., 2004). The cross-correlation coefficient results are listed in Supplementary file 1. The geometry of the PBS–PSII supercomplex was measured with the ‘distance’ and ‘angles’ command in UCSF Chimera. To analyze the arrangement of the PBS–PSII supercomplex in the thylakoid membrane, the double PBS–PSII and PBS–PSII sub-tomogram averaging maps were re-mapped back into the tomograms with the refined orientations and positions taking use of home-made scripts. The segmentation of the thylakoid membrane was performed with TomoSegMemTV (Martinez-Sanchez et al., 2014), Amira software (FEI Visualization Sciences Group), and UCSF ChimeraX (Pettersen et al., 2021). Segmentation of the subunits of the double PBS–PSII and PBS–PSII supercomplex was conducted in UCSF Chimera with the ‘Segmentation panel’. The densities of the subunits were then extracted by subtracting other parts with the subtract function. Surface rendering was further colored with the color zone panel.

Data availability

The cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under the accession number EMD-31241, EMD-31242, EMD-31243, EMD-31244, EMD-31245.

The following data sets were generated
    1. Sui S-F
    2. X M Li
    3. M J Li
    4. Ma JF
    (2021) Electron Microscopy Data Bank
    ID EMD-31241. Structure of the PBS-PSII supercomplex from red algae.
    1. Sui S-F
    2. X M Li
    3. M J Li
    4. Ma JF
    (2021) Electron Microscopy Data Bank
    ID EMD-31242. Structure of the double phycobilisome-photosystem II supercomplex from red alga.
    1. Sui S-F
    2. X M Li
    3. M J Li
    4. Ma JF
    (2021) Electron Microscopy Data Bank
    ID EMD-31243. Cryo-electron tomography of phycobilisome-photosystem II supercomplex on the thylakoid.
    1. Sui S-F
    2. X M Li
    3. M J Li
    4. Ma JF
    (2021) Electron Microscopy Data Bank
    ID EMD-31244. Cryo-electron tomography of phycobilisome-photosystem II supercomplex on the thylakoid.
    1. Sui S-F
    2. X M Li
    3. M J Li
    4. Ma JF
    (2021) Electron Microscopy Data Bank
    ID EMD-31245. Cryo-electron tomography of phycobilisome-photosystem II supercomplex on the thylakoid.
The following previously published data sets were used
    1. Ma JF
    2. You X
    3. Sun S
    4. Wang XX
    5. Qin S
    6. Sui S-F
    (2020) RCSB Protein Data Bank
    ID 6KGX. Structure of the phycobilisome from the red alga Porphyridium purpureum.

References

    1. Capuano V
    2. Braux AS
    3. Tandeau de Marsac N
    4. Houmard J
    (1991)
    The “anchor polypeptide” of cyanobacterial phycobilisomes Molecular characterization of the Synechococcus sp PCC 6301 apce gene
    The Journal of Biological Chemistry 266:7239–7247.
    1. Su X
    2. Fraenkel PG
    3. Bogorad L
    (1992)
    Excitation energy transfer from phycocyanin to chlorophyll in an apcA-defective mutant of Synechocystis sp. PCC 6803
    The Journal of Biological Chemistry 267:22944–22950.
  1. Book
    1. Zhao J
    2. Zhou J
    3. Bryant DA
    (1992)
    Energy transfer processes in phycobilisomes as deduced from analyses of mutants of Synechococcus sp. PCC7002
    In: Murata N, editors. Research in Photosynthesis, Vol 1.. Kluwer Academic Publishers. pp. 25–32.

Decision letter

  1. Andrew P Carter
    Reviewing Editor; MRC Laboratory of Molecular Biology, United Kingdom
  2. Volker Dötsch
    Senior Editor; Goethe University, Germany
  3. Conrad W Mullineaux
    Reviewer; Queen Mary University of London, United Kingdom

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Interactions between reaction centres and phycobilisome light-harvesting complexes is crucial for photosynthesis in cyanobacteria and red algae, which between them account for a major fraction of photosynthesis on earth. This work uses in situ cryo-electron tomography to understand those interactions which were previously not amenable to study because they are too labile to survive isolation of the complexes.

Decision letter after peer review:

Thank you for submitting your article "In situcryo-ET structure of phycobilisome-photosystem II supercomplex from red alga" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Volker Dötsch as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Conrad W Mullineaux (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) The reviewers were overall excited by the insights your in-situ cryo-ET analysis gave into the organization of the PBS/PSII complex. However in some places we felt the descriptions of atom level interactions were not justified by the data. In other regions the descriptions are speculative but worth including.

Please remove the following discussions:

– Page 4 lines 35-36. The claim of a 45 deg rotation is unsupported by the data and should be removed.

– Page 7, lines 1-11. This section is too speculative and conclusions are drawn regarding missing residue of a loop which are really not supported by the data.

Please modify other parts of the manuscript to make it clear that the descriptions are more speculative:

– Page 7, lines 18-20.

– Page 8, lines 5-13.

– Page 8, lines 20-24.

2) The methods section needs more detail to allow readers to understand how the structure were solved. Please see the comments by reviewer 2 below.

Reviewer #1:

This manuscript reports the in-situ structure of the red algal phycobilisome while still bound to the thylakoid membrane. It is a follow-up to the same group's impressive cryo EM structure of the isolated phycobilisome from the same organism (Ma et al. Nature 2020). While the resolution of the current structure is significantly lower than that of the isolated complex, (13+ A vs 2.8 A), it has the advantage of revealing the organization in the membrane, the connections to Photosystem II and additional details of the structure that may be too labile to survive the isolation of the complex.

Overall, I think this is an impressive study. Of particular note is the discovery of the Type C PSII, which I don't think has been previously reported. The emphasis on the excitation energy coupling pathways to PSII is also important new information, although the relatively low resolution of the current structure makes that analysis less certain.

I suggest that the authors emphasize in clearer terms that this is an in-situ structure that is complementary to the structure of the isolated complex and reveals elements of the in vivo organization that can never be obtained through structures of the isolated complex, no matter how high the resolution. I think some readers may not appreciate that point sufficiently, and the current manuscript does not emphasize this critical point.

Reviewer #2:

Li et al., report the in situ structure of the phycobilisome (PBS) – photosystem II (PSII) complex in P. purpureum cells. They prepare cells by cryo-FIB/SEM, and collect cryo-tomograms around thylakoid membrane regions that are decorated by PBS/PSII regular arrays. They then perform subtomogram averaging of PBS pairs and single complexes to obtain structures at resolutions ranging between 13 and 16 A. These allow fitting of available atomic models and analysis of the molecular architecture and interfaces that characterise the complex.

The authors are able to confirm the overall architecture of PBS which was obtained from previous studies, but also reveal a number of novel features, including unexpected stoichiometry of PBS:PSII, and some uncharacterised interfaces. The results are organised into three main sections:

– Overall structure of the PBS-PSII supercomplex

Here the authors describe how each unit is composed of a PBS and three PSII dimers, two of which contact PBS directly, and two seem connected through some novel densities (here called 'lateral densities'), which are assigned to extra PBS hexamer. This was not reported before and it might be an interface that is stable only in an in situ context. This section is strong and the conclusions are well justified.

– Interaction pattern between PBS and PSII

Here the authors utilise the fitted atomic models to analyse interfaces between PBS and the PSII dimers at the molecular level. While the resolution of their subtomogram averaging map is sufficient for rigid body fitting of the relevant structures, I find the analysis here too speculative, particularly when it comes to analysis of subdomains (i.e. the PB-loop and the 33 missing residues, and the 117deg rotation of CP43 N-terminus).

The claim of two pathways for electron transfer and their description is unsupported by the data.

– Supplementary interactions intermediated by three connector proteins

Here the authors identify regions of density that cannot be explained with the fitted atomic models, and cautiously assign them to connecting proteins. They then analyse the positions of these connecting proteins and describe the interfaces they bridge. While the discovery of these new densities and the overall description of their contacts is convincing, I feel that again the authors draw conclusions at a level of molecular detail that is too speculative given the resolution.

Overall this is a novel and solid paper with regards to the overall description of PBS/PSII in situ, its stoichiometry and general architecture, which should be of interest to the fields of electron transport and in situ cryo-tomography. However some of the claims are not justified by the data, in particular those that analyse interactions in molecular detail.

Comments and recommendations below in no particular order

– While overall the paper is well written and logically explained, there are several typographical and grammatical errors. Moreover, certain sections are unclear and rephrasing could improve legibility significantly. For example: Page 4, Lines 19-23, Page 5, Lines 27-30, Page 6, Lines 22-32, Page 7 Line 16.

– It is not clear from the methods if the maps were post-processed in any way, and low-pass filtered at the stated resolution. They should be filtered so as to remove the noisy features visible in the figures.

– Red boxes in Figure supplement 1B,C: it would be useful to see what is included also along the z direction.

– Page 4 lines 35-36. The claim of a 45 deg rotation is unsupported by the data and should be removed.

– I found the nomenclature in Figure 2A a bit confusing, would it not be easier to call subunits A1,A2, B1,B2,C1,C2, A'1,A'2,[…],C'1,C'2?

– As far as I understand, Figurer 2 shows two different maps in panel A and B. This should be clearly stated in the legend.

– For general readers some introduction to PBS architecture is required: what are Rod a, Rod b, etc?

– Page 6, lines 28-32. It is unclear whether the authors claim or not that the extra hexamer is a PE hexamer, as there are some contradictory sentences.

– There are a series of unsupported claims:

– Page 7, lines 1-11. This section is too speculative and conclusions are drawn regarding missing residue of a loop which are really not supported by the data.

– Page 7, lines 18-20. This sentence is not supported by the data. There is also no description in the methods of how the 117 deg angle was derived (flexible fitting?).

– Page 8, lines 5-13. As above

– Page 8, lines 20-24. As above

I suggest the authors limit themselves to a general description of which domain interact with each other rather than going into sub-domain or even residue-level considerations. There is value in their description of the architecture and interfaces, if only it would be made in less detailed terms.

– Page 7, lines 24-28. The basis for the claim are unclear.

– Re: connector proteins, how were they identified? By difference mapping with the fitted atomic model? This needs to be stated clearly.

– Many details are missing in the methods (at least those parts I can understand: tomogram collection and subtomogram abveraging). What was the total dose across the tilt series (or tilt increment)? There is no reference at all to ctf: was it corrected (I assume so within relion)? How was it estimated? Was dose damage compensation performed? Was the dataset divided in two halves according to the gold standard? In consequence, are the FSC reported gold standard FSCs?

– The authors need to add the FSC (and possibly local resolution estimations) as supplementary figures.

Reviewer #3:

The authors were able to convincingly identify a phycobilisome-photosystem II complex in their cryo-electron tomograms of chloroplasts from a red alga, and to obtain high-resolution structural models by sub-tomogram averaging and fitting parts of known structures to the tomographic data. A crucial feature of work is that it reports structures in situ, in intact flash-frozen sections. That's important for 2 reasons. Firstly, the in situ data reveals the larger-scale organisation of the complexes in the membrane, which is crucially important for photosynthetic performance. Secondly, the association between phycobilisomes and reaction centres appears rather plastic and is highly unstable (at least in vitro), raising the possibility of significant artefacts in any preparation of isolated complexes. So, in situ information of this sort is really needed to provide convincing answers. I am not well qualified to judge the technical aspects of the tomographic analysis, but it all looks sound as far as I am able to judge. The manuscript is very well-written and well-presented, and there is an excellent, balanced discussion of the findings.

https://doi.org/10.7554/eLife.69635.sa1

Author response

Essential revisions:

1) The reviewers were overall excited by the insights your in-situ cryo-ET analysis gave into the organization of the PBS/PSII complex. However in some places we felt the descriptions of atom level interactions were not justified by the data. In other regions the descriptions are speculative but worth including.

Please remove the following discussions:

– Page 4 lines 35-36. The claim of a 45 deg rotation is unsupported by the data and should be removed.

– Page 7, lines 1-11. This section is too speculative and conclusions are drawn regarding missing residue of a loop which are really not supported by the data.

Please modify other parts of the manuscript to make it clear that the descriptions are more speculative:

– Page 7, lines 18-20.

– Page 8, lines 5-13.

– Page 8, lines 20-24.

2) The methods section needs more detail to allow readers to understand how the structure were solved. Please see the comments by reviewer 2 below.

Thank you for your precious comments and summary. We have deleted the discussions not supported by the data (P. 4 lines 35-36; P. 7 lines 1-11) in the revised manuscript. According to reviewers ' constructive suggestions, we have also rewritten the speculative descriptions, which have been explained in the point-by-point responses to reviewers' comments. In the revised manuscript, all the experimental details suggested by reviewers have been included.

Reviewer #1:

This manuscript reports the in-situ structure of the red algal phycobilisome while still bound to the thylakoid membrane. It is a follow-up to the same group's impressive cryo EM structure of the isolated phycobilisome from the same organism (Ma et al. Nature 2020). While the resolution of the current structure is significantly lower than that of the isolated complex, (13+ A vs 2.8 A), it has the advantage of revealing the organization in the membrane, the connections to Photosystem II and additional details of the structure that may be too labile to survive the isolation of the complex.

Overall, I think this is an impressive study. Of particular note is the discovery of the Type C PSII, which I don't think has been previously reported. The emphasis on the excitation energy coupling pathways to PSII is also important new information, although the relatively low resolution of the current structure makes that analysis less certain.

I suggest that the authors emphasize in clearer terms that this is an in-situ structure that is complementary to the structure of the isolated complex and reveals elements of the in vivo organization that can never be obtained through structures of the isolated complex, no matter how high the resolution. I think some readers may not appreciate that point sufficiently, and the current manuscript does not emphasize this critical point.

We appreciate your insightful suggestions. We have incorporated your suggestions into the last paragraph of the introduction part (Page 4, lines 9-12, lines 16-17).

Reviewer #2:

Li et al., report the in situ structure of the phycobilisome (PBS) – photosystem II (PSII) complex in P. purpureum cells. They prepare cells by cryo-FIB/SEM, and collect cryo-tomograms around thylakoid membrane regions that are decorated by PBS/PSII regular arrays. They then perform subtomogram averaging of PBS pairs and single complexes to obtain structures at resolutions ranging between 13 and 16 A. These allow fitting of available atomic models and analysis of the molecular architecture and interfaces that characterise the complex.

The authors are able to confirm the overall architecture of PBS which was obtained from previous studies, but also reveal a number of novel features, including unexpected stoichiometry of PBS:PSII, and some uncharacterised interfaces. The results are organised into three main sections:

– Overall structure of the PBS-PSII supercomplex

Here the authors describe how each unit is composed of a PBS and three PSII dimers, two of which contact PBS directly, and two seem connected through some novel densities (here called 'lateral densities'), which are assigned to extra PBS hexamer. This was not reported before and it might be an interface that is stable only in an in situ context. This section is strong and the conclusions are well justified.

– Interaction pattern between PBS and PSII

Here the authors utilise the fitted atomic models to analyse interfaces between PBS and the PSII dimers at the molecular level. While the resolution of their subtomogram averaging map is sufficient for rigid body fitting of the relevant structures, I find the analysis here too speculative, particularly when it comes to analysis of subdomains (i.e. the PB-loop and the 33 missing residues, and the 117deg rotation of CP43 N-terminus).

The claim of two pathways for electron transfer and their description is unsupported by the data.

– Supplementary interactions intermediated by three connector proteins

Here the authors identify regions of density that cannot be explained with the fitted atomic models, and cautiously assign them to connecting proteins. They then analyse the positions of these connecting proteins and describe the interfaces they bridge. While the discovery of these new densities and the overall description of their contacts is convincing, I feel that again the authors draw conclusions at a level of molecular detail that is too speculative given the resolution.

Overall this is a novel and solid paper with regards to the overall description of PBS/PSII in situ, its stoichiometry and general architecture, which should be of interest to the fields of electron transport and in situ cryo-tomography. However some of the claims are not justified by the data, in particular those that analyse interactions in molecular detail.

Thank you for your precious comments. We have carefully considered your valuable

suggestions and comments. We have dealt with your precious suggestions and responded to each comment. As a summary, we have re-analyzed the interaction interfaces between PBS and PSII at the domain level in the revised manuscript with the new figures and videos, as suggested by you. The interfaces provide convincing evidence for the multiple energy transfer.

We have clarified how we found the new densities and discussed their connections with PBS and PSII at a domain level. The claims that are not justified by the data have been removed.

Comments and recommendations below in no particular order

– While overall the paper is well written and logically explained, there are several typographical and grammatical errors. Moreover, certain sections are unclear and rephrasing could improve legibility significantly. For example: Page 4, Lines 19-23, Page 5, Lines 27-30, Page 6, Lines 22-32, Page 7 Line 16.

We appreciate your insightful suggestions. We have rewritten these unclear descriptions.

– It is not clear from the methods if the maps were post-processed in any way, and low-pass filtered at the stated resolution. They should be filtered so as to remove the noisy features visible in the figures.

Thanks for noticing this. The previous PBS-PSII supercomplex map at a resolution of 13.2 Å is post-processed with the program implemented in Relion software. To evade the noisy features, we have updated all the figures with the non-post-processing map at 14.3 Å resolution, which does not present noisy features. We have provided a new Figure 2—figure supplement 1 to show the resolution estimation and local resolution maps for the double PBS-PSII supercomplex and PBS-PSII supercomplex.

– Red boxes in Figure supplement 1B,C: it would be useful to see what is included also along the z direction.

We appreciate your insightful suggestions. We have updated Figure 1—figure supplement 1B, 1C with the Z-direction project. Moreover, we have provided two related movies (Video 2 corresponding to panel B and Video 3 corresponding to panel C) to show the sequential slices back and forth in Z-direction through the tomographic volume. To provide a clear three dimensional view of the tomographic volumes we used in the manuscript, we provided Video 1 (Corresponding to Figure 1A), Video 5 (Corresponding to Figure 6B).

– Page 4 lines 35-36. The claim of a 45 deg rotation is unsupported by the data and should be removed.

We appreciate your insightful suggestions. We have removed the discussion.

– I found the nomenclature in Figure 2A a bit confusing, would it not be easier to call subunits A1,A2, B1,B2,C1,C2, A'1,A'2,[…],C'1,C'2?

We appreciate your insightful suggestions. We have replaced the annotations throughout the revised manuscript and the corresponding figures.

– As far as I understand, Figurer 2 shows two different maps in panel A and B. This should be clearly stated in the legend.

Thank you for noting this. We have rewritten the legend as follows:

“(A) The density map of double PBS-PSII supercomplex at a resolution of 15.6 Å, presented in two perpendicular views. The center-to-center distance of two adjacent PBSs is approximately 345 Å. Two random circles indicated by black or red dashed lines mark the six PSII monomers binding with each of the two PBSs. PBS1 is associated with PSII monomer A1, A2, B1, B2, C1, and C'2. PBS2 connects with A'1, A'2, B'1, B'2, C'1, C’’2. The surface threshold is 0.09. (B) The density map of the PBS-PSII supercomplex at a resolution of 14.3 Å fitted with the single particle model of PBS (EMDB code EMD-9976, PDB code 6KGX) and X-ray structure of PSII (PDB code 4YUU), presented in two perpendicular views. The lateral hexamer was fitted with the single-particle model of the Rod a distal PE hexamer. The surface threshold is 0.059. (C) The magnified image shows that the two PsbO subunits bind with each other at the interface of the adjacent PSII dimers (Inset). A map of PSII dimers A and B, segmented from (B) with the same surface threshold level. The arrow indicates the binding site.”

– For general readers some introduction to PBS architecture is required: what are Rod a, Rod b, etc?

We appreciate your insightful suggestions. We have included the introduction about Rod a, Rod b, PBS core, PSII composition in the introduction section as follows:

“The cryo-EM structure of PBS in red alga Porphyridium purpureum (Ma et al., 2020) shows that the PBS consists of a tricylindrical PBS core,14 rods (Rod a – Rod g, Rod a’– Rod g’), 8 individual extra PE (αβ)6 hexamers (Ha – Hd, Ha’– Hd’) and 24 individual extra PE α or β subunits. The core contains one top cylinder B, composed of two allophycocyanin (APC) (αβ)3 trimers, and two bottom cylinders (A and A’), each of which is assembled by three APC trimers (A1-A3, A2 and A3 form APC hexamer). The rods consist of phycoerythrin (PE) and phycocyanin (PC) hexamers or only PE hexamers. For example, Rod a is composited of one basal PC hexamer and two distal PE hexamers. Thus, the energy absorbed by Rod transfers uni-directionally from the distal PE to the basal PC, and then funnels to APC in the core, and eventually to the two terminal emitters, including chromophores in the core–membrane linker protein (LCM, also called ApcE) (Capuano et al., 1991; Lundell et al., 1981; Tang et al., 2015) and allophycocyanin D (ApcD) (Glazer and Bryant, 1975; Peng et al., 2014).”

(Page 2, lines 32 - 36, Page 3, lines 1-7).

"The main subunits of PSII include the reaction center D1, D2 protein, and chlorophyll-a binding proteins, CP43 and CP47. Both CP43 and CP47 subunits have been reported to mediate the energy transfer from PBS to the reaction center (Ueno et al., 2017)."

(Page 3, lines 16-19).

– Page 6, lines 28-32. It is unclear whether the authors claim or not that the extra hexamer is a PE hexamer, as there are some contradictory sentences.

We appreciate your insightful suggestions. We have rewritten the unclear description. In the revised manuscript, we claimed the lateral hexamer might be a PE hexamer as follows:

“To analyze the lateral densities on both sides of PBS connecting PBS to PSII dimer C, we

further improved the resolution of the PBS-PSII supercomplex to 13.2 Å after post-processing from the 14.3 Å resolution map (Figure 2—figure supplement 1D). The slice view of the PBSPSII map shows that the densities are round discs with stronger densities in the center (Figure 2—figure supplement 3E–G). These features are reminiscent of the structure of the extra hexamer found in the single-particle cryo-EM structure of P. purpureum PBS. Moreover, the lateral densities are of the same size and shape as the extra hexamer. As all eight extra hexamers in P. purpureum PBS are PE hexamers, we hypothesize that the lateral density could be contributed by a PE hexamer, which is referred to as the lateral hexamer (Figure 2B). Further analysis indicated that the lateral hexamer connects with the bottom hexamer of Rod a and the second hexamer of Rod e, which are the PC and PE hexamers, respectively (Figure 2—figure supplement 3E).” (Page 6, lines 34 -36, Page 7, lines 1-8).

– There are a series of unsupported claims:

– Page 7, lines 1-11. This section is too speculative and conclusions are drawn regarding missing residue of a loop which are really not supported by the data.

– Page 7, lines 18-20. This sentence is not supported by the data. There is also no description in the methods of how the 117 deg angle was derived (flexible fitting?).

– Page 8, lines 5-13. As above

– Page 8, lines 20-24. As above

I suggest the authors limit themselves to a general description of which domain interact with each other rather than going into sub-domain or even residue-level considerations. There is value in their description of the architecture and interfaces, if only it would be made in less detailed terms.

Thank you for noting this. We have removed the inappropriate descriptions and re-analyzed the interaction at the domain level by rephrasing the whole section 'Interaction pattern between PBS and PSII' (Page 7, lines 12-28) with the new figures (Figure 3, Figure 3—figure supplement 1). We do not paste the text here.

In addition, we have rewritten the unclear description of Page 8, lines 5-13, and lines 20-24. The revised parts are rephrased on Page 8, lines 6-23 with new figures (Figure 4). We do not paste the text here.

– Page 7, lines 24-28. The basis for the claim are unclear.

Thank you for noting this. We think that the claim is too speculative and we deleted this

claim. According to the reviewer’s suggestion, we have rephrased the whole section of

“Interaction pattern between PBS and PSII” with a general description of subunit interaction. (Page 7, lines 12-28)

– Re: connector proteins, how were they identified? By difference mapping with the fitted atomic model? This needs to be stated clearly.

We appreciate your insightful suggestions. Yes, we identified the connector proteins by

difference mapping with the fitted atomic models. We have rewritten the unclear description as follows:

“After docking the atomic models of PBS, PSII dimer and two lateral hexamers into the density map of PBS-PSII supercomplex, we still observed three extra densities that are not occupied by any model. Two of them are associated with the PBS and PSII (Figure 4—figure supplement 1 A), and the third one is associated with the lateral hexamer and PSII (Figure 4—figure supplement 1B). Since we could not identify the proteins corresponding to these densities, we temporarily deemed them "connectors" (connector 1 – 3; Figure 4, Figure 4—figure supplement 2A–E).” (Page 7, lines 32-35)

– Many details are missing in the methods (at least those parts I can understand: tomogram collection and subtomogram abveraging). What was the total dose across the tilt series (or tilt increment)? There is no reference at all to ctf: was it corrected (I assume so within relion)? How was it estimated? Was dose damage compensation performed? Was the dataset divided in two halves according to the gold standard? In consequence, are the FSC reported gold standard FSCs?

We appreciate your insightful suggestions. We have included the missed details in the

method section as follows.

The tomographic tilt series were recorded from -50° to +66° with an increment of 2° using the unidirectional strategy. (Page 13, lines 23-24)

CTF correction was estimated with CTFFIND4 (Rohou and Grigorieff, 2015) implemented in RELION. Dose damage compensation was performed with the script provided in the RELION tutorial. (Page 13, lines 38-39, Page 14, line 1)

During the auto-refinement, the datasets were automatically divided into two halves by RELION. (Page 14, lines 11-12)

– The authors need to add the FSC (and possibly local resolution estimations) as supplementary figures.

We appreciate your insightful suggestions. We have provided a new supplementary figure, Figure 2—figure supplement 1.

https://doi.org/10.7554/eLife.69635.sa2

Article and author information

Author details

  1. Meijing Li

    Key Laboratory for Protein Sciences of Ministry of Education, Beijing Advanced Innovation Center for Structural Biology & Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing, China
    Present address
    Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, Martinsried, Germany
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft
    Contributed equally with
    Jianfei Ma
    Competing interests
    None
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3931-7905
  2. Jianfei Ma

    State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology & Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing, China
    Contribution
    Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Validation, Visualization, Writing – original draft
    Contributed equally with
    Meijing Li
    Competing interests
    none
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2016-2280
  3. Xueming Li

    Key Laboratory for Protein Sciences of Ministry of Education, Beijing Advanced Innovation Center for Structural Biology & Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing, China
    Contribution
    Formal analysis, Funding acquisition, Project administration, Supervision, Validation, Writing - review and editing, designed project, designed project
    For correspondence
    lixueming@mail.tsinghua.edu.cn
    Competing interests
    None
  4. Sen-Fang Sui

    1. State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology & Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing, China
    2. Department of Biology, Southern University of Science and Technology, Guangdong, China
    Contribution
    Formal analysis, Funding acquisition, Project administration, Supervision, Validation, Writing - review and editing, designed project, designed project
    For correspondence
    suisf@mail.tsinghua.edu.cn
    Competing interests
    None
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5253-2592

Funding

National Basic Research Program (2016YFA0501101)

  • Sen-Fang Sui

National Basic Research Program (2017YFA0504600)

  • Sen-Fang Sui

National Basic Research Program (2016YFA0501102)

  • Xueming Li

National Basic Research Program (2016YFA0501902)

  • Xueming Li

National Natural Science Foundation of China (31670745)

  • Sen-Fang Sui

National Natural Science Foundation of China (31861143048)

  • Sen-Fang Sui

National Natural Science Foundation of China (31722015)

  • Xueming Li

National Natural Science Foundation of China (32000848)

  • Jianfei Ma

Advanced Innovation Center for Structural Biology

  • Xueming Li

Tsinghua-Peking Joint Center for Life Sciences

  • Xueming Li

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was supported by the National Basic Research Program (2016YFA0501101 and 2017YFA0504600 to S-FS, 2016YFA0501102 and 2016YFA0501902 to XL), the National Natural Science Foundation of China (31670745 and 31861143048 to S-FS, 31722015 to XL, 32000848 to JM), Advanced Innovation Center for Structural Biology (to XL), Tsinghua-Peking Joint Center for Life Sciences (to XL). We thank the staff at the Tsinghua University Branch of the National Protein Science Facility (Beijing) for their technical support on the Cryo-EM and High-Performance Computation platforms. We thank XM Li for participating in facility supports. We thank JL Lei for data collection; J Liu and S Li for their recommendations on computation; YC Wang for recommendations on phylogenetic analysis; S K Cheppali, XC Qin, S Sun, and Y N Xiao for comments on the manuscript.

Senior Editor

  1. Volker Dötsch, Goethe University, Germany

Reviewing Editor

  1. Andrew P Carter, MRC Laboratory of Molecular Biology, United Kingdom

Reviewer

  1. Conrad W Mullineaux, Queen Mary University of London, United Kingdom

Publication history

  1. Received: April 21, 2021
  2. Accepted: August 19, 2021
  3. Version of Record published: September 13, 2021 (version 1)

Copyright

© 2021, Li et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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