Characterization of a Giant PSI Supercomplex in the Symbiotic Dinoflagellate Symbiodiniaceae.

Symbiodiniaceae are symbiotic dinoflagellates that provide photosynthetic products to corals. Because corals are distributed across a wide range of depths in the ocean, Symbiodiniaceae species must adapt to various light environments to optimize their photosynthetic performance. However, as few biochemical studies of Symbiodiniaceae photosystems have been reported, the molecular mechanisms of photoadaptation in this algal family remain poorly understood. Here, to investigate the photosynthetic machineries in Symbiodiniaceae, we purified and characterized the PSI supercomplex from the genome-sequenced Breviolum minutum (formerly Symbiodinium minutum). Mass spectrometry analysis revealed 25 light-harvesting complexes (LHCs), including both LHCF and LHCR families, from the purified PSI-LHC supercomplex. Single-particle electron microscopy showed unique giant supercomplex structures of PSI that were associated with the LHCs. Moreover, the PSI-LHC supercomplex contained a significant amount of the xanthophyll cycle pigment diadinoxanthin. Upon high light treatment, B. minutum cells showed increased nonphotochemical quenching, which was correlated with the conversion of diadinoxanthin to diatoxanthin, occurring preferentially in the PSI-LHC supercomplex. The possible role of PSI-LHC in photoprotection in Symbiodiniaceae is discussed.

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

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.

Ten antenna proteins are associated with the core in the supramolecular organization of the photosystem I supercomplex in Chlamydomonas reinhardtii.

Photosystem I (PSI) is a large pigment-protein complex mediating light-driven charge separation and generating a highly negative redox potential, which is eventually utilized to produce organic matter. In plants and algae, PSI possesses outer antennae, termed light-harvesting complex I (LHCI), which increase the energy flux to the reaction center. The number of outer antennae for PSI in the green alga Chlamydomonas reinhardtii is known to be larger than that of land plants. However, their exact number and location remain to be elucidated. Here, applying a newly established sample purification procedure, we isolated a highly pure PSI-LHCI supercomplex containing all nine LHCA gene products under state 1 conditions. Single-particle cryo-EM revealed the 3D structure of this supercomplex at 6.9 Å resolution, in which the densities near the PsaF and PsaJ subunits were assigned to two layers of LHCI belts containing eight LHCIs, whereas the densities between the PsaG and PsaH subunits on the opposite side of the LHCI belt were assigned to two extra LHCIs. Using single-particle cryo-EM, we also determined the 2D projection map of the lhca2 mutant, which confirmed the assignment of LHCA2 and LHCA9 to the densities between PsaG and PsaH. Spectroscopic measurements of the PSI-LHCI supercomplex suggested that the bound LHCA2 and LHCA9 proteins have the ability to increase the light-harvesting energy for PSI. We conclude that the PSI in C. reinhardtii has a larger and more distinct outer-antenna organization and higher light-harvesting capability than that in land plants.

Cryo-electron Microscopy of Protein Cages.

Protein cages are one of the most widely studied objects in the field of cryogenic electron microscopy-encompassing natural and synthetic constructs, from enzymes assisting protein folding such as chaperonin to virus capsids. Tremendous diversity of morphology and function is demonstrated by the structure and role of proteins, some of which are nearly ubiquitous, while others are present in few organisms. Protein cages are often highly symmetrical, which helps improve the resolution obtained by cryo-electron microscopy (cryo-EM). Cryo-EM is the study of vitrified samples using an electron probe to image the subject. A sample is rapidly frozen in a thin layer on a porous grid, attempting to keep the sample as close to a native state as possible. This grid is kept at cryogenic temperatures throughout imaging in an electron microscope. Once image acquisition is complete, a variety of software packages may be employed to carry out analysis and reconstruction of three-dimensional structures from the two-dimensional micrograph images. Cryo-EM can be used on samples that are too large or too heterogeneous to be amenable to other structural biology techniques like NMR or X-ray crystallography. In recent years, advances in both hardware and software have provided significant improvements to the results obtained using cryo-EM, recently demonstrating true atomic resolution from vitrified aqueous samples. Here, we review these advances in cryo-EM, especially in that of protein cages, and introduce several tips for situations we have experienced.

Six states of Enterococcus hirae V-type ATPase reveals non-uniform rotor rotation during turnover.

The vacuolar-type ATPase from Enterococcus hirae (EhV-ATPase) is a thus-far unique adaptation of V-ATPases, as it performs Na+ transport and demonstrates an off-axis rotor assembly. Recent single molecule studies of the isolated V1 domain have indicated that there are subpauses within the three major states of the pseudo three-fold symmetric rotary enzyme. However, there was no structural evidence for these. Herein we activate the EhV-ATPase complex with ATP and identified multiple structures consisting of a total of six states of this complex by using cryo-electron microscopy. The orientations of the rotor complex during turnover, especially in the intermediates, are not as perfectly uniform as expected. The densities in the nucleotide binding pockets in the V1 domain indicate the different catalytic conditions for the six conformations. The off-axis rotor and its' interactions with the stator a-subunit during rotation suggests that this non-uniform rotor rotation is performed through the entire complex.

A novel capsid protein network allows the characteristic internal membrane structure of Marseilleviridae giant viruses.

Marseilleviridae is a family of giant viruses, showing a characteristic internal membrane with extrusions underneath the icosahedral vertices. However, such large objects, with a maximum diameter of 250 nm are technically difficult to examine at sub-nanometre resolution by cryo-electron microscopy. Here, we tested the utility of 1 MV high-voltage cryo-EM (cryo-HVEM) for single particle structural analysis (SPA) of giant viruses using tokyovirus, a species of Marseilleviridae, and revealed the capsid structure at 7.7 Å resolution. The capsid enclosing the viral DNA consisted primarily of four layers: (1) major capsid proteins (MCPs) and penton proteins, (2) minor capsid proteins (mCPs), (3) scaffold protein components (ScPCs), and (4) internal membrane. The mCPs showed a novel capsid lattice consisting of eight protein components. ScPCs connecting the icosahedral vertices supported the formation of the membrane extrusions, and possibly act like tape measure proteins reported in other giant viruses. The density on top of the MCP trimer was suggested to include glycoproteins. This is the first attempt at cryo-HVEM SPA. We found the primary limitations to be the lack of automated data acquisition and software support for collection and processing and thus achievable resolution. However, the results pave the way for using cryo-HVEM for structural analysis of larger biological specimens.

Cryo-electron microscopy of the giant viruses.

High-resolution study of the giant viruses presents one of the latest challenges in cryo-electron microscopy (EM) of viruses. Too small for light microscopy but too large for easy study at high resolution by EM, they range in size from ∼0.2 to 2 µm from high-symmetry icosahedral viruses, such as Paramecium burseria Chlorella virus 1, to asymmetric forms like Tupanvirus or Pithovirus. To attain high resolution, two strategies exist to study these large viruses by cryo-EM: first, increasing the acceleration voltage of the electron microscope to improve sample penetration and overcome the limitations imposed by electro-optical physics at lower voltages, and, second, the method of 'block-based reconstruction' pioneered by Michael G. Rossmann and his collaborators, which resolves the latter limitation through an elegant leveraging of high symmetry but cannot overcome sample penetration limitations. In addition, more recent advances in both computational capacity and image processing also yield assistance in studying the giant viruses. Especially, the inclusion of Ewald sphere correction can provide large improvements in attainable resolutions for 300 kV electron microscopes. Despite this, the study of giant viruses remains a significant challenge.

Below 3 Å structure of apoferritin using a multipurpose TEM with a side entry cryoholder.

Recently, the structural analysis of protein complexes by cryo-electron microscopy (cryo-EM) single particle analysis (SPA) has had great impact as a biophysical method. Many results of cryo-EM SPA are based on data acquired on state-of-the-art cryo-electron microscopes customized for SPA. These are currently only available in limited locations around the world, where securing machine time is highly competitive. One potential solution for this time-competitive situation is to reuse existing multi-purpose equipment, although this comes with performance limitations. Here, a multi-purpose TEM with a side entry cryo-holder was used to evaluate the potential of high-resolution SPA, resulting in a 3 Å resolution map of apoferritin with local resolution extending to 2.6 Å. This map clearly showed two positions of an aromatic side chain. Further, examination of optimal imaging conditions depending on two different multi-purpose electron microscope and camera combinations was carried out, demonstrating that higher magnifications are not always necessary or desirable. Since automation is effectively a requirement for large-scale data collection, and augmenting the multi-purpose equipment is possible, we expanded testing by acquiring data with SerialEM using a ß-galactosidase test sample. This study demonstrates the possibilities of more widely available and established electron microscopes, and their applications for cryo-EM SPA.

Supramolecular tholos-like architecture constituted by archaeal proteins without functional annotation.

Euryarchaeal genomes encode proteasome-assembling chaperone homologs, PbaA and PbaB, although archaeal proteasome formation is a chaperone-independent process. Homotetrameric PbaB functions as a proteasome activator, while PbaA forms a homopentamer that does not interact with the proteasome. Notably, PbaA forms a complex with PF0014, an archaeal protein without functional annotation. In this study, based on our previous research on PbaA crystal structure, we performed an integrative analysis of the supramolecular structure of the PbaA/PF0014 complex using native mass spectrometry, solution scattering, high-speed atomic force microscopy, and electron microscopy. The results indicated that this highly thermostable complex constitutes ten PbaA and ten PF0014 molecules, which are assembled into a dumbbell-shaped structure. Two PbaA homopentameric rings correspond to the dumbbell plates, with their N-termini located outside of the plates and C-terminal segments left mobile. Furthermore, mutant PbaA lacking the mobile C-terminal segment retained the ability to form a complex with PF0014, allowing 3D modeling of the complex. The complex shows a five-column tholos-like architecture, in which each column comprises homodimeric PF0014, harboring a central cavity, which can potentially accommodate biomacromolecules including proteins. Our findings provide insight into the functional roles of Pba family proteins, offering a novel framework for designing functional protein cages.

Amphipol-assisted purification method for the highly active and stable photosystem II supercomplex of Chlamydomonas reinhardtii.

Photosystem II (PSII) splits water and drives electron transfer to plastoquinone via photochemical reactions using light energy. It is surrounded by light-harvesting complex II (LHCII) to form the PSII-LHCII supercomplex. Complete characterization of its structure and function has, however, been hampered due to instability of the complex in the presence of detergent. To overcome this problem, we developed a new procedure for purifying the PSII-LHCII supercomplexes of Chlamydomonas reinhardtii employing amphipol A8-35. The obtained supercomplexes showed little LHCII dissociation even 4 days after purification. Oxygen-evolving activity was retained within amphipol if the extrinsic polypeptides were kept associated by betaine. Electron microscopy revealed that this method also improved structural uniformity and that the major organization was C2 S2 M2 L2 .