Structural determination of the large photosystem II-light-harvesting complex II supercomplex of Chlamydomonas reinhardtii using nonionic amphipol

Abstract

In photosynthetic organisms, photosystem II (PSII) is a large membrane protein complex, consisting of a pair of core complexes surrounded by an array of variable numbers of light-harvesting complex (LHC) II proteins. Previously reported structures of the PSII-LHCII supercomplex of the green alga Chlamydomonas reinhardtii exhibit significant structural heterogeneity, but recently improved purification methods employing ionic amphipol A8-35 have enhanced supercomplex stability, providing opportunities for determining a more intact structure. Herein, we present a 5.8 Å cryo-EM map of the C. reinhardtii PSII-LHCII supercomplex containing six LHCII trimers (C₂S₂M₂L₂). Utilizing a newly developed nonionic amphipol-based purification and stabilizing method, we purified the largest photosynthetic supercomplex to the highest percentage of the intact configuration reported to date. We found that the interprotein distances within the light-harvesting complex array in the green algal photosystem are larger than those previously observed in higher plants, indicating that the potential route of energy transfer in the PSII-LHCII supercomplex in green algae may be altered. Interestingly, we also observed an asymmetric PSII-LHCII supercomplex structure comprising C₂S₂M₁L₁ in the same sample. Moreover, we found a new density adjacent to the PSII core complex, attributable to a single-transmembrane helix. It was previously unreported in the cryo-EM maps of PSII-LHCII supercomplexes from land plants.

Keywords: Chlamydomonas reinhardtii; algae; cryo-electron microscopy; light-harvesting complex (antenna complex); photosynthesis; single-particle analysis.

Figure 1.

A, SDG ultracentrifugation and SDS-PAGE of the NAPol-stabilized and α-DDM–solubilized PSII–LHCII supercomplexes. SDG of the two samples were performed on the same run. The specific bands of the PSII–LHCII supercomplexes (boxed in dashed red) were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue R-250. The identities of polypeptides were verified through MS analysis on the in-gel protease digestion products of the excised SDS-PAGE bands. B, example cryo-micrograph of NAPol-stabilized C. reinhardtii PSII–LHCII, raw (upper panel) with autopicked particles circled in red (lower panel). Scale bar, 100 nm, C, schematic diagram of PSII–LHCII array in C. reinhardtii, perpendicular to the membrane. D1, PsbA; D2, PsbD; 43, PsbC; 47, PsbB; 26, CP26; 29, CP29; S, LHCII S-trimer; M, LHCII M-trimer; L, LHCII L-trimer; fuchsia circle, PsbTc; green ring, PsbL; red ring, PsbM; I, PsbI; W, PsbW; H, PsbH; X, PsbX; F, PsbF; E, PsbE; J, PsbJ; K, PsbK; Z, PsbZ. The question mark indicates new density tentatively assigned to Ycf12. The dashed black line indicates dimer symmetry.

Figure 2.

2D and 3D views of the C₂S₂M₂L₂-type C. reinhardtii PSII–LHCII supercomplex. A, 2D reprojection of the 3D reconstruction (this report) of PSII–LHCII supercomplex perpendicular to the thylakoid membrane (from the luminal side), B, the same as A, rotated 90° on the y axis. C, perpendicular view of A, rotated 90° on the x axis. D, translucent view of the 3D reconstruction of the PSII–LHCII supercomplex perpendicular to the thylakoid membrane with a rigid-body fit model based on PDB code 5XNL (from the luminal side). E, the same as D, rotated 90° on the y axis. F, perpendicular view of D, rotated 90° on the x axis. The PSII core, minor LHCs, and S-trimers are between 5 and 6 Å resolution. M- and L-trimers are more flexible and thus have a lower resolution. Maps were contoured to 3σ (D–F). Maps colored by local resolution can be found in Fig. S3. The assignments of the LHCII trimers follow the nomenclature used in Boekema et al. (5) and further by Tokutsu et al. (10). Red arrows, NAPol ring density. Scale bars, 10 nm.

Figure 3.

2D reprojection of the C. reinhardtii PSII–LHCII cryo-EM reconstruction with PDB models of other species PSII–LHCII super complexes overlaid. A, fully aligned model of the C. reinhardtii PSII–LHCII including all LHCII trimers using PDB code 5XNL (3). B, PDB code 3JCU (18) overlaid against C. reinhardtii PSII–LHCII. C, PDB code 5MDX (17) overlaid against C. reinhardtii PSII–LHCII. The lack of edge-middle core density is attributed to missing PsbP and PsbQ in 5MDX (17). D, PDB code 5XNL (3) overlaid against C. reinhardtii PSII–LHCII. E–H, close-up views of one side of the core/LHCII interface of A–D, respectively. For all published models and maps of PSII–LHCII from land plants, the core regions align well, whereas the LHCII array is compressed relative to the C. reinhardtii LHCII array. The white arrows indicate PDB edge. This also highlights the different angle of M trimer between C₂S₂M₂L₂ and C₂S₂M₂ type PSII–LHCII. Scale bars, 10 nm in A–D and 5 nm in E–H.

Figure 4.

Novel transmembrane density alongside the core dimer, which we putatively ascribe to Ycf12 (orange). A, the area containing this previously unreported density is circled in red, 2D reprojection. The contrast has been increased 70%. B, view from the luminal side of PSII–LHCII super complex (the same orientation as the 2D reprojection of A). C, view parallel to the membrane, turned on x and y axes by 90° with NAPol ring erased for clarity. The red arrow in A indicates the direction of view toward the Ycf12 location in C. This novel density appears to coordinate to PsbJ, PsbK, and PsbZ with PsbE and PsbF (pale purple) to the right. CP26 is in mustard yellow. This density is present on both sides of the core complexes for both the C₂S₂M₂L₂ and C₂S₂M₁L₁ PSII–LHCII supercomplexes. Scale bar, 10 nm.

Figure 5.

Representative 2D projections of the three subspecies of PSII–LHCII supercomplex identified within this study. A, symmetric C₂S₂M₂L₂ type with six LHCII trimers. Particle count is 19,864. B, asymmetric C₂S₂M₁L₁ type with four LHCII trimers. Particle count is 9035. C, symmetric C₂S₂ type with two LHCII trimers. Particle count is 371. The percentages of the three subspecies stabilized with NAPol are in the bottom right for each particle type. Scale bar, 10 nm.