In situ structural determination of cyanobacterial phycobilisome-PSII supercomplex by STAgSPA strategy.

Photosynthesis converting solar energy to chemical energy is one of the most important chemical reactions on earth. In cyanobacteria, light energy is captured by antenna system phycobilisomes (PBSs) and transferred to photosynthetic reaction centers of photosystem II (PSII) and photosystem I (PSI). While most of the protein complexes involved in photosynthesis have been characterized by in vitro structural analyses, how these protein complexes function together in vivo is not well understood. Here we implemented STAgSPA, an in situ structural analysis strategy, to solve the native structure of PBS-PSII supercomplex from the cyanobacteria Arthrospira sp. FACHB439 at resolution of ~3.5 Å. The structure reveals coupling details among adjacent PBSs and PSII dimers, and the collaborative energy transfer mechanism mediated by multiple super-PBS in cyanobacteria. Our results provide insights into the diversity of photosynthesis-related systems between prokaryotic cyanobacteria and eukaryotic red algae but are also a methodological demonstration for high-resolution structural analysis in cellular or tissue samples.

Structure of the red-shifted Fittonia albivenis photosystem I.

Photosystem I (PSI) from Fittonia albivenis, an Acanthaceae ornamental plant, is notable among green plants for its red-shifted emission spectrum. Here, we solved the structure of a PSI-light harvesting complex I (LHCI) supercomplex from F. albivenis at 2.46-Å resolution using cryo-electron microscopy. The supercomplex contains a core complex of 14 subunits and an LHCI belt with four antenna subunits (Lhca1-4) similar to previously reported angiosperm PSI-LHCI structures; however, Lhca3 differs in three regions surrounding a dimer of low-energy chlorophylls (Chls) termed red Chls, which absorb far-red beyond visible light. The unique amino acid sequences within these regions are exclusively shared by plants with strongly red-shifted fluorescence emission, suggesting candidate structural elements for regulating the energy state of red Chls. These results provide a structural basis for unraveling the mechanisms of light harvest and transfer in PSI-LHCI of under canopy plants and for designing Lhc to harness longer-wavelength light in the far-red spectral range.

Structure of the intact Tom20 receptor in the human translocase of the outer membrane complex.

The translocase of the outer membrane (TOM) complex serves as the main gate for preproteins entering mitochondria and thus plays a pivotal role in sustaining mitochondrial stability. Precursor proteins, featuring amino-terminal targeting signals (presequences) or internal targeting signals, are recognized by the TOM complex receptors Tom20, Tom22, and Tom70, and then translocated into mitochondria through Tom40. By using chemical cross-linking to stabilize Tom20 in the TOM complex, this study unveils the structure of the human TOM holo complex, encompassing the intact Tom20 component, at a resolution of approximately 6 Å by cryo-electron microscopy. Our structure shows the TOM holo complex containing only one Tom20 subunit, which is located right at the center of the complex and stabilized by extensive interactions with Tom22, Tom40, and Tom6. Based on the structure, we proposed a possible translocation mode of TOM complex, by which different receptors could work simultaneously to ensure that the preproteins recognized by them are all efficiently translocated into the mitochondria.

Activity-based metaproteomics driven discovery and enzymological characterization of potential α-galactosidases in the mouse gut microbiome.

The gut microbiota offers an extensive resource of enzymes, but many remain uncharacterized. To distinguish the activities of similar annotated proteins and mine the potentially applicable ones in the microbiome, we applied an effective Activity-Based Metaproteomics (ABMP) strategy using a specific activity-based probe (ABP) to screen the entire gut microbiome for directly discovering active enzymes and their potential applications, not for exploring host-microbiome interactions. By using an activity-based cyclophellitol aziridine probe specific to α-galactosidases (AGAL), we successfully identified and characterized several gut microbiota enzymes possessing AGAL activities. Cryo-electron microscopy analysis of a newly characterized enzyme (AGLA5) revealed the covalent binding conformations between the AGAL5 active site and the cyclophellitol aziridine ABP, which could provide insights into the enzyme's catalytic mechanism. The four newly characterized AGALs have diverse potential activities, including raffinose family oligosaccharides (RFOs) hydrolysis and enzymatic blood group transformation. Collectively, we present a ABMP platform that facilitates gut microbiota AGALs discovery, biochemical activity annotations and potential industrial or biopharmaceutical applications.

Ultrastructural insights into cellular organization, energy storage and ribosomal dynamics of an ammonia-oxidizing archaeon from oligotrophic oceans.

Introduction: Nitrososphaeria, formerly known as Thaumarchaeota, constitute a diverse and widespread group of ammonia-oxidizing archaea (AOA) inhabiting ubiquitously in marine and terrestrial environments, playing a pivotal role in global nitrogen cycling. Despite their importance in Earth's ecosystems, the cellular organization of AOA remains largely unexplored, leading to a significant unanswered question of how the machinery of these organisms underpins metabolic functions. Methods: In this study, we combined spherical-chromatic-aberration-corrected cryo-electron tomography (cryo-ET), scanning transmission electron microscopy (STEM), and energy dispersive X-ray spectroscopy (EDS) to unveil the cellular organization and elemental composition of Nitrosopumilus maritimus SCM1, a representative member of marine Nitrososphaeria. Results and Discussion: Our tomograms show the native ultrastructural morphology of SCM1 and one to several dense storage granules in the cytoplasm. STEM-EDS analysis identifies two types of storage granules: one type is possibly composed of polyphosphate and the other polyhydroxyalkanoate. With precise measurements using cryo-ET, we observed low quantity and density of ribosomes in SCM1 cells, which are in alignment with the documented slow growth of AOA in laboratory cultures. Collectively, these findings provide visual evidence supporting the resilience of AOA in the vast oligotrophic marine environment.