Structural insight into synergistic activation of human 3-methylcrotonyl-CoA carboxylase.

The enzymes 3-methylcrotonyl-coenzyme A (CoA) carboxylase (MCC), pyruvate carboxylase and propionyl-CoA carboxylase belong to the biotin-dependent carboxylase family located in mitochondria. They participate in various metabolic pathways in human such as amino acid metabolism and tricarboxylic acid cycle. Many human diseases are caused by mutations in those enzymes but their structures have not been fully resolved so far. Here we report an optimized purification strategy to obtain high-resolution structures of intact human endogenous MCC, propionyl-CoA carboxylase and pyruvate carboxylase in different conformational states. We also determine the structures of MCC bound to different substrates. Analysis of MCC structures in different states reveals the mechanism of the substrate-induced, multi-element synergistic activation of MCC. These results provide important insights into the catalytic mechanism of the biotin-dependent carboxylase family and are of great value for the development of new drugs for the treatment of related diseases.

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.

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.

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.

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.

Regulatory dynamics of the higher-plant PSI-LHCI supercomplex during state transitions.

State transition is a fundamental light acclimation mechanism of photosynthetic organisms in response to the environmental light conditions. This process rebalances the excitation energy between photosystem I (PSI) and photosystem II through regulated reversible binding of the light-harvesting complex II (LHCII) to PSI. However, the structural reorganization of PSI-LHCI, the dynamic binding of LHCII, and the regulatory mechanisms underlying state transitions are less understood in higher plants. In this study, using cryoelectron microscopy we resolved the structures of PSI-LHCI in both state 1 (PSI-LHCI-ST1) and state 2 (PSI-LHCI-LHCII-ST2) from Arabidopsis thaliana. Combined genetic and functional analyses revealed novel contacts between Lhcb1 and PsaK that further enhanced the binding of the LHCII trimer to the PSI core with the known interactions between phosphorylated Lhcb2 and the PsaL/PsaH/PsaO subunits. Specifically, PsaO was absent in the PSI-LHCI-ST1 supercomplex but present in the PSI-LHCI-LHCII-ST2 supercomplex, in which the PsaL/PsaK/PsaA subunits undergo several conformational changes to strengthen the binding of PsaO in ST2. Furthermore, the PSI-LHCI module adopts a more compact configuration with shorter Mg-to-Mg distances between the chlorophylls, which may enhance the energy transfer efficiency from the peripheral antenna to the PSI core in ST2. Collectively, our work provides novel structural and functional insights into the mechanisms of light acclimation during state transitions in higher plants.

The structural basis for light acclimation in phycobilisome light harvesting systems systems in Porphyridium purpureum.

Photosynthetic organisms adapt to changing light conditions by manipulating their light harvesting complexes. Biophysical, biochemical, physiological and genetic aspects of these processes are studied extensively. The structural basis for these studies is lacking. In this study we address this gap in knowledge by focusing on phycobilisomes (PBS), which are large structures found in cyanobacteria and red algae. In this study we focus on the phycobilisomes (PBS), which are large structures found in cyanobacteria and red algae. Specifically, we examine red algae (Porphyridium purpureum) grown under a low light intensity (LL) and a medium light intensity (ML). Using cryo-electron microscopy, we resolve the structure of ML-PBS and compare it to the LL-PBS structure. The ML-PBS is 13.6 MDa, while the LL-PBS is larger (14.7 MDa). The LL-PBS structure have a higher number of closely coupled chromophore pairs, potentially the source of the red shifted fluorescence emission from LL-PBS. Interestingly, these differences do not significantly affect fluorescence kinetics parameters. This indicates that PBS systems can maintain similar fluorescence quantum yields despite an increase in LL-PBS chromophore numbers. These findings provide a structural basis to the processes by which photosynthetic organisms adapt to changing light conditions.

Nuclear export of pre-60S particles through the nuclear pore complex.

The nuclear pore complex (NPC) is the bidirectional gate that mediates the exchange of macromolecules or their assemblies between nucleus and cytoplasm1-3. The assembly intermediates of the ribosomal subunits, pre-60S and pre-40S particles, are among the largest cargoes of the NPC and the export of these gigantic ribonucleoproteins requires numerous export factors4,5. Here we report the cryo-electron microscopy structure of native pre-60S particles trapped in the channel of yeast NPCs. In addition to known assembly factors, multiple factors with export functions are also included in the structure. These factors in general bind to either the flexible regions or subunit interface of the pre-60S particle, and virtually form many anchor sites for NPC binding. Through interactions with phenylalanine-glycine (FG) repeats from various nucleoporins of NPC, these factors collectively facilitate the passage of the pre-60S particle through the central FG repeat network of the NPC. Moreover, in silico analysis of the axial and radial distribution of pre-60S particles within the NPC shows that a single NPC can take up to four pre-60S particles simultaneously, and pre-60S particles are enriched in the inner ring regions close to the wall of the NPC with the solvent-exposed surface facing the centre of the nuclear pore. Our data suggest a translocation model for the export of pre-60S particles through the NPC.

Structural insights into assembly of TRAPPII and its activation of Rab11/Ypt32.

Transport protein particle (TRAPP) complexes belong to the multisubunit tethering complex. They are guanine nucleotide exchange factors (GEFs) that play essential roles in secretory and endocytic recycling pathway and autophagy. There are two major forms of TRAPP complexes, TRAPPII and TRAPPIII, which share a core set of small subunits. TRAPPIII activates Rab1, while TRAPPII primarily activates Rab11. A steric gating mechanism has been proposed to control the substrate selection in vivo. However, the detailed mechanisms underlying the transition from TRAPPIII's GEF activity for Rab1 to TRAPPII's GEF activity for Rab11 and the roles of the complex-specific subunits in this transition are insufficiently understood. In this review, we discuss recent advances in understanding the mechanism of specific activation of Rab11/Ypt32 by TRAPPII, with a particular focus on new findings from structural studies.

Determining protein structures in cellular lamella at pseudo-atomic resolution by GisSPA.

Cryo-electron tomography is a major tool used to study the structure of protein complexes in situ. However, the throughput of tilt-series image data collection is still quite low. Here, we show that GisSPA, a GPU accelerated program, can translationally and rotationally localize the target protein complex in cellular lamellae, as prepared with a focused ion beam, using single cryo-electron microscopy images without tilt-series, and reconstruct the protein complex at near-atomic resolution. GisSPA allows high-throughput data collection without the acquisition of tilt-series images and reconstruction of the tomogram, which is essential for high-resolution reconstruction of asymmetric or low-symmetry protein complexes. We demonstrate the power of GisSPA with 3.4-Å and 3.9-Å resolutions of resolving phycobilisome and tetrameric photosystem II complex structures in cellular lamellae, respectively. In this work, we present GisSPA as a practical tool that facilitates high-resolution in situ protein structure determination.

In situ structure of the red algal phycobilisome-PSII-PSI-LHC megacomplex.

In oxygenic photosynthetic organisms, light energy is captured by antenna systems and transferred to photosystem II (PSII) and photosystem I (PSI) to drive photosynthesis1,2. The antenna systems of red algae consist of soluble phycobilisomes (PBSs) and transmembrane light-harvesting complexes (LHCs)3. Excitation energy transfer pathways from PBS to photosystems remain unclear owing to the lack of structural information. Here we present in situ structures of PBS-PSII-PSI-LHC megacomplexes from the red alga Porphyridium purpureum at near-atomic resolution using cryogenic electron tomography and in situ single-particle analysis4, providing interaction details between PBS, PSII and PSI. The structures reveal several unidentified and incomplete proteins and their roles in the assembly of the megacomplex, as well as a huge and sophisticated pigment network. This work provides a solid structural basis for unravelling the mechanisms of PBS-PSII-PSI-LHC megacomplex assembly, efficient energy transfer from PBS to the two photosystems, and regulation of energy distribution between PSII and PSI.

Structure of the Acidobacteria homodimeric reaction center bound with cytochrome c.

Photosynthesis converts light energy to chemical energy to fuel life on earth. Light energy is harvested by antenna pigments and transferred to reaction centers (RCs) to drive the electron transfer (ET) reactions. Here, we present cryo-electron microscopy (cryo-EM) structures of two forms of the RC from the microaerophilic Chloracidobacterium thermophilum (CabRC): one containing 10 subunits, including two different cytochromes; and the other possessing two additional subunits, PscB and PscZ. The larger form contained 2 Zn-bacteriochlorophylls, 16 bacteriochlorophylls, 10 chlorophylls, 2 lycopenes, 2 hemes, 3 Fe4S4 clusters, 12 lipids, 2 Ca2+ ions and 6 water molecules, revealing a type I RC with an ET chain involving two hemes and a hybrid antenna containing bacteriochlorophylls and chlorophylls. Our results provide a structural basis for understanding the excitation energy and ET within the CabRC and offer evolutionary insights into the origin and adaptation of photosynthetic RCs.

Structural insights into PA3488-mediated inactivation of Pseudomonas aeruginosa PldA.

PldA, a phospholipase D (PLD) effector, catalyzes hydrolysis of the phosphodiester bonds of glycerophospholipids-the main component of cell membranes-and assists the invasion of the opportunistic pathogen Pseudomonas aeruginosa. As a cognate immunity protein, PA3488 can inhibit the activity of PldA to avoid self-toxicity. However, the precise inhibitory mechanism remains elusive. We determine the crystal structures of full-length and truncated PldA and the cryogenic electron microscopy structure of the PldA-PA3488 complex. Structural analysis reveals that there are different intermediates of PldA between the "open" and "closed" states of the catalytic pocket, accompanied by significant conformational changes in the "lid" region and the peripheral helical domain. Through structure-based mutational analysis, we identify the key residues responsible for the enzymatic activity of PldA. Together, these data provide an insight into the molecular mechanisms of PldA invasion and its neutralization by PA3488, aiding future design of PLD-targeted inhibitors and drugs.

Structural basis of Tom20 and Tom22 cytosolic domains as the human TOM complex receptors.

Mitochondrial preproteins synthesized in cytosol are imported into mitochondria by a multisubunit translocase of the outer membrane (TOM) complex. Functioned as the receptor, the TOM complex components, Tom 20, Tom22, and Tom70, recognize the presequence and further guide the protein translocation. Their deficiency has been linked with neurodegenerative diseases and cardiac pathology. Although several structures of the TOM complex have been reported by cryoelectron microscopy (cryo-EM), how Tom22 and Tom20 function as TOM receptors remains elusive. Here we determined the structure of TOM core complex at 2.53 Å and captured the structure of the TOM complex containing Tom22 and Tom20 cytosolic domains at 3.74 Å. Structural analysis indicates that Tom20 and Tom22 share a similar three-helix bundle structural feature in the cytosolic domain. Further structure-guided biochemical analysis reveals that the Tom22 cytosolic domain is responsible for binding to the presequence, and the helix H1 is critical for this binding. Altogether, our results provide insights into the functional mechanism of the TOM complex recognizing and transferring preproteins across the mitochondrial membrane.

Near-atomic structure of the inner ring of the Saccharomyces cerevisiae nuclear pore complex.

Nuclear pore complexes (NPCs) mediate bidirectional nucleocytoplasmic transport of substances in eukaryotic cells. However, the accurate molecular arrangement of NPCs remains enigmatic owing to their huge size and highly dynamic nature. Here we determined the structure of the asymmetric unit of the inner ring (IR monomer) at 3.73 Å resolution by single-particle cryo-electron microscopy, and created an atomic model of the intact IR consisting of 192 molecules of 8 nucleoporins. In each IR monomer, the Z-shaped Nup188-Nup192 complex in the middle layer is sandwiched by two approximately parallel rhomboidal structures in the inner and outer layers, while Nup188, Nup192 and Nic96 link all subunits to constitute a relatively stable IR monomer. In contrast, the intact IR is assembled by loose and instable interactions between IR monomers. These structures, together with previously reported structural information of IR, reveal two distinct interaction modes between IR monomers and extensive flexible connections in IR assembly, providing a structural basis for the stability and malleability of IR.

Structural basis for assembly of TRAPPII complex and specific activation of GTPase Ypt31/32.

Transport protein particle (TRAPP) complexes belong to the multiprotein tethering complex and exist in three forms-core TRAPP/TRAPPI, TRAPPII, and TRAPPIII. TRAPPII activates GTPase Ypt31/Ypt32 as the guanine nucleotide exchange factor in the trans-Golgi network to determine the maturation of Golgi cisternae into post-Golgi carriers in yeast. Here, we present cryo-EM structures of yeast TRAPPII in apo and Ypt32-bound states. All the structures show a dimeric architecture assembled by two triangle-shaped monomers, while the monomer in the apo state exhibits both open and closed conformations, and the monomer in the Ypt32-bound form only captures the closed conformation. Located in the interior of the monomer, Ypt32 binds with both core TRAPP/TRAPPI and Trs120 via its nucleotide-binding domain and binds with Trs31 via its hypervariable domain. Combined with functional analysis, the structures provide insights into the assembly of TRAPPII and the mechanism of the specific activation of Ypt31/Ypt32 by TRAPPII.

Brain structure and structural basis of neurodegenerative diseases.

The brain is one of the most complex organs in nature. In this organ, multiple neurons, neuron clusters, or multiple brain regions are interconnected to form a complex structural network where various brain functions are completed through interaction. In recent years, multiple tools and techniques have been developed to analyze the composition of different cell types in the brain and to construct the brain atlas on macroscopic, mesoscopic, and microscopic levels. Meanwhile, researchers have found that many neuropsychiatric diseases, such as Parkinson's disease, Alzheimer's disease, and Huntington's disease, are closely related to abnormal changes of brain structure, which means the investigation in brain structure not only provides a new idea for understanding the pathological mechanism of the diseases, but also provides imaging markers for early diagnosis and potential treatment. This article pays attention to the research of human brain structure, reviews the research progress of human brain structure and the structural mechanism of neurodegenerative diseases, and discusses the problems and prospects in the field.

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

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.