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.

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.

Structural basis of energy transfer in Porphyridium purpureum phycobilisome.

Photosynthetic organisms have developed various light-harvesting systems to adapt to their environments1. Phycobilisomes are large light-harvesting protein complexes found in cyanobacteria and red algae2-4, although how the energies of the chromophores within these complexes are modulated by their environment is unclear. Here we report the cryo-electron microscopy structure of a 14.7-megadalton phycobilisome with a hemiellipsoidal shape from the red alga Porphyridium purpureum. Within this complex we determine the structures of 706 protein subunits, including 528 phycoerythrin, 72 phycocyanin, 46 allophycocyanin and 60 linker proteins. In addition, 1,598 chromophores are resolved comprising 1,430 phycoerythrobilin, 48 phycourobilin and 120 phycocyanobilin molecules. The markedly improved resolution of our structure compared with that of the phycobilisome of Griffithsia pacifica5 enabled us to build an accurate atomic model of the P. purpureum phycobilisome system. The model reveals how the linker proteins affect the microenvironment of the chromophores, and suggests that interactions of the aromatic amino acids of the linker proteins with the chromophores may be a key factor in fine-tuning the energy states of the chromophores to ensure the efficient unidirectional transfer of energy.

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.

Structural insights into a dimeric Psb27-photosystem II complex from a cyanobacterium Thermosynechococcus vulcanus.

Photosystem II (PSII) is a multisubunit pigment-protein complex and catalyzes light-driven water oxidation, leading to the conversion of light energy into chemical energy and the release of molecular oxygen. Psb27 is a small thylakoid lumen-localized protein known to serve as an assembly factor for the biogenesis and repair of the PSII complex. The exact location and binding fashion of Psb27 in the intermediate PSII remain elusive. Here, we report the structure of a dimeric Psb27-PSII complex purified from a psbV deletion mutant (ΔPsbV) of the cyanobacterium Thermosynechococcus vulcanus, solved by cryo-electron microscopy. Our structure showed that Psb27 is associated with CP43 at the luminal side, with specific interactions formed between Helix 2 and Helix 3 of Psb27 and a loop region between Helix 3 and Helix 4 of CP43 (loop C) as well as the large, lumen-exposed and hydrophilic E-loop of CP43. The binding of Psb27 imposes some conflicts with the N-terminal region of PsbO and also induces some conformational changes in CP43, CP47, and D2. This makes PsbO unable to bind in the Psb27-PSII. Conformational changes also occurred in D1, PsbE, PsbF, and PsbZ; this, together with the conformational changes occurred in CP43, CP47, and D2, may prevent the binding of PsbU and induce dissociation of PsbJ. This structural information provides important insights into the regulation mechanism of Psb27 in the biogenesis and repair of PSII.

Structure of phycobilisome from the red alga Griffithsia pacifica.

Life on Earth depends on photosynthesis for its conversion of solar energy to chemical energy. Photosynthetic organisms have developed a variety of light-harvesting systems to capture sunlight. The largest light-harvesting complex is the phycobilisome (PBS), the main light-harvesting antenna in cyanobacteria and red algae. It is composed of phycobiliproteins and linker proteins but the assembly mechanisms and energy transfer pathways of the PBS are not well understood. Here we report the structure of a 16.8-megadalton PBS from a red alga at 3.5 Å resolution obtained by single-particle cryo-electron microscopy. We modelled 862 protein subunits, including 4 linkers in the core, 16 rod-core linkers and 52 rod linkers, and located a total of 2,048 chromophores. This structure reveals the mechanisms underlying specific interactions between linkers and phycobiliproteins, and the formation of linker skeletons. These results provide a firm structural basis for our understanding of complex assembly and the mechanisms of energy transfer within the PBS.

Synaptotagmin-7 is a key factor for bipolar-like behavioral abnormalities in mice.

The pathogenesis of bipolar disorder (BD) has remained enigmatic, largely because genetic animal models based on identified susceptible genes have often failed to show core symptoms of spontaneous mood cycling. However, pedigree and induced pluripotent stem cell (iPSC)-based analyses have implicated that dysfunction in some key signaling cascades might be crucial for the disease pathogenesis in a subpopulation of BD patients. We hypothesized that the behavioral abnormalities of patients and the comorbid metabolic abnormalities might share some identical molecular mechanism. Hence, we investigated the expression of insulin/synapse dually functioning genes in neurons derived from the iPSCs of BD patients and the behavioral phenotype of mice with these genes silenced in the hippocampus. By these means, we identified synaptotagmin-7 (Syt7) as a candidate risk factor for behavioral abnormalities. We then investigated Syt7 knockout (KO) mice and observed nocturnal manic-like and diurnal depressive-like behavioral fluctuations in a majority of these animals, analogous to the mood cycling symptoms of BD. We treated the Syt7 KO mice with clinical BD drugs including olanzapine and lithium, and found that the drug treatments could efficiently regulate the behavioral abnormalities of the Syt7 KO mice. To further verify whether Syt7 deficits existed in BD patients, we investigated the plasma samples of 20 BD patients and found that the Syt7 mRNA level was significantly attenuated in the patient plasma compared to the healthy controls. We therefore concluded that Syt7 is likely a key factor for the bipolar-like behavioral abnormalities.

Unique organization of photosystem I-light-harvesting supercomplex revealed by cryo-EM from a red alga.

Photosystem I (PSI) is one of the two photosystems present in oxygenic photosynthetic organisms and functions to harvest and convert light energy into chemical energy in photosynthesis. In eukaryotic algae and higher plants, PSI consists of a core surrounded by variable species and numbers of light-harvesting complex (LHC)I proteins, forming a PSI-LHCI supercomplex. Here, we report cryo-EM structures of PSI-LHCR from the red alga Cyanidioschyzon merolae in two forms, one with three Lhcr subunits attached to the side, similar to that of higher plants, and the other with two additional Lhcr subunits attached to the opposite side, indicating an ancient form of PSI-LHCI. Furthermore, the red algal PSI core showed features of both cyanobacterial and higher plant PSI, suggesting an intermediate type during evolution from prokaryotes to eukaryotes. The structure of PsaO, existing in eukaryotic organisms, was identified in the PSI core and binds three chlorophylls a and may be important in harvesting energy and in mediating energy transfer from LHCII to the PSI core under state-2 conditions. Individual attaching sites of LHCRs with the core subunits were identified, and each Lhcr was found to contain 11 to 13 chlorophylls a and 5 zeaxanthins, which are apparently different from those of LHCs in plant PSI-LHCI. Together, our results reveal unique energy transfer pathways different from those of higher plant PSI-LHCI, its adaptation to the changing environment, and the possible changes of PSI-LHCI during evolution from prokaryotes to eukaryotes.

Expression and purification of functional PDGF receptor beta.

Platelet Derived Growth Factor receptors (PDGFRs), members of receptor tyrosine kinase superfamily, play essential roles in early hematopoiesis, angiogenesis and organ development. Dysregulation of PDGF receptor signaling under pathological conditions associates with cancers, vascular diseases, and fibrotic diseases. Therefore, they are attractive targets in drug development. Like any other membrane proteins with a single-pass transmembrane domain, the high-resolution structural information of the full-length PDGF receptors is still not resolved. It is caused, at least in part, by the technical challenges in the expression and purification of the functional, full-length PDGF receptors. Herein, we reported our experimental details in expression and purification of the full-length PDGFRß from mammalian cells. We found that purified PDGFRß remained in two different oligomeric states, presumably the monomer and the dimer, with basal kinase activity in detergent micelles. Addition of PDGF-B promoted dimerization and elevated kinase activity of the receptor, suggesting that purified receptors were functional.

Molecular basis for the formation of ribonucleoprotein complex of Crimean-Congo hemorrhagic fever virus.

Negative-sense single-strand RNA (-ssRNA) viruses comprise a large family of pathogens that cause severe human infectious diseases. All -ssRNA viruses encode a nucleocapsid protein (NP) to encapsidate the viral genome, which, together with polymerase, forms a ribonucleoprotein complex (RNP) that is packaged into virions and acts as the template for viral replication and transcription. In our previous work, we solved the monomeric structure of NP encoded by Crimean-Congo hemorrhagic fever virus (CCHFV), which belongs to the Nairovirus genus within the Bunyaviridae family, and revealed its unusual endonuclease activity. However, the mechanism of CCHFV RNP formation remains unclear, due to the difficulty in reconstructing the oligomeric CCHFV NP-RNA complex. Here, we identified and isolated the oligomeric CCHFV NP-RNA complex that formed in expression cells. Sequencing of RNA extracted from the complex revealed sequence specificity and suggested a potential encapsidation signal facilitating the association between NP and viral genome. A cryo-EM reconstruction revealed the ring-shaped architecture of the CCHFV NP-RNA oligomer, thus defining the interaction between the head and stalk domains that results in NP multimerization. This structure also suggested a modified gating mechanism for viral genome encapsidation, in which both the head and stalk domains participate in RNA binding. This work provides insight into the distinct mechanism underlying CCHFV RNP formation compared to other -ssRNA viruses.

Bunyamwera virus possesses a distinct nucleocapsid protein to facilitate genome encapsidation.

Bunyamwera virus (BUNV), which belongs to the genus Orthobunyavirus, is the prototypical virus of the Bunyaviridae family. Similar to other negative-sense single-stranded RNA viruses, bunyaviruses possess a nucleocapsid protein (NP) to facilitate genomic RNA encapsidation and virus replication. The structures of two NPs of members of different genera within the Bunyaviridae family have been reported. However, their structures, RNA-binding features, and functions beyond RNA binding significantly differ from one another. Here, we report the crystal structure of the BUNV NP-RNA complex. The polypeptide of the BUNV NP was found to possess a distinct fold among viral NPs. An N-terminal arm and a C-terminal tail were found to interact with neighboring NP protomers to form a tetrameric ring-shaped organization. Each protomer bound a 10-nt RNA molecule, which was acquired from the expression host, in the positively charged crevice between the N and C lobes. Inhomogeneous oligomerization was observed for the recombinant BUNV NP-RNA complex, which was similar to the Rift Valley fever virus NP-RNA complex. This result suggested that the flexibility of one NP protomer with adjacent protomers underlies the BUNV ribonucleoprotein complex (RNP) formation. Electron microscopy revealed that the monomer-sized NP-RNA complex was the building block of the natural BUNV RNP. Combined with previous results indicating that mutagenesis of the interprotomer or protein-RNA interface affects BUNV replication, our structure provides a great potential for understanding the mechanism underlying negative-sense single-stranded RNA RNP formation and enables the development of antiviral therapies targeting BUNV RNP formation.

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.

Reconstitution of functionally efficient SecA-dependent protein-conducting channels: transformation of low-affinity SecA-liposome channels to high-affinity SecA-SecYEG-SecDF·YajC channels.

Previous work showed that SecA alone can promote protein translocation and ion-channel activity in liposomes, and that SecYEG increases efficiency as well as signal peptide specificity. We now report that SecDF·YajC further increases translocation and ion-channel activity. These activities of reconstituted SecA-SecYEG-SecDF·YajC-liposome are almost the same as those of native membranes, indicating the transformation of reconstituted functional high-affinity protein-conducting channels from the low-affinity SecA-channels.

Specificity of SecYEG for PhoA precursors and SecA homologs on SecA protein-conducting channels.

Previous studies showed that Escherichia coli membranes depleted of SecYEG are capable of translocating certain precursor proteins, but not other precursors such as pPhoA, indicating a differential requirement for SecYEG. In this study, we examined the role of SecYEG in pPhoA translocation using a purified reconstituted SecA-liposomes system. We found that translocation of pPhoA, in contrast to that of pOmpA, requires the presence of purified SecYEG. A differential specificity of the SecYEG was also revealed in its interaction with SecA: EcSecYEG did not enhance SecA-mediated pOmpA translocation by purified SecA either from Pseudomonas aeruginosa or Bacillus subtilis. Neither was SecYEG required for eliciting ion channel activity, which could be opened by unfolded pPhoA or unfolded PhoA. Addition of the SecYEG complex did restore the specificity of signal peptide recognition in the ion-channel activity. We concluded that SecYEG confers specificity in interacting with protein precursors and SecAs.

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.

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.

Cellular internalization of exosomes occurs through phagocytosis.

Exosomes play important roles in many physiological and pathological processes. However, the exosome-cell interaction mode and the intracellular trafficking pathway of exosomes in their recipient cells remain unclear. Here, we report that exosomes derived from K562 or MT4 cells are internalized more efficiently by phagocytes than by non-phagocytic cells. Most exosomes were observed attached to the plasma membrane of non-phagocytic cells, while in phagocytic cells these exosomes were found to enter via phagocytosis. Specifically, they moved to phagosomes together with phagocytic polystyrene carboxylate-modified latex beads (biospheres) and were further sorted into phagolysosomes. Moreover, exosome internalization was dependent on the actin cytoskeleton and phosphatidylinositol 3-kinase, and could be inhibited by the knockdown of dynamin2 or overexpression of a dominant-negative form of dynamin2. Further, antibody pretreatment assays demonstrated that tim4 but not tim1 was involved in exosomes uptake. We also found that exosomes did not enter the internalization pathway involving caveolae, macropinocytosis and clathrin-coated vesicles. Our observation that the cellular uptake of exosomes occurs through phagocytosis has important implications for exosome-cell interactions and the exosome intracellular trafficking pathway.

SecA alone can promote protein translocation and ion channel activity: SecYEG increases efficiency and signal peptide specificity.

SecA is an essential component of the Sec-dependent protein translocation pathway across cytoplasmic membranes in bacteria. Escherichia coli SecA binds to cytoplasmic membranes at SecYEG high affinity sites and at phospholipid low affinity sites. It has been widely viewed that SecYEG functions as the essential protein-conducting channel through which precursors cross the membranes in bacterial Sec-dependent pathways, and that SecA functions as a motor to hydrolyze ATP in translocating precursors through SecYEG channels. We have now found that SecA alone can promote precursor translocation into phospholiposomes. Moreover, SecA-liposomes elicit ionic currents in Xenopus oocytes. Patch-clamp recordings further show that SecA alone promotes signal peptide- or precursor-dependent single channel activity. These activities were observed with the functional SecA at about 1-2 µM. The results show that SecA alone is sufficient to promote protein translocation into liposomes and to elicit ionic channel activity at the phospholipids low affinity binding sites, thus indicating that SecA is able to form the protein-conducting channels. Even so, such SecA-liposomes are less efficient than those with a full complement of Sec proteins, and lose the signal-peptide proofreading function, resembling the effects of PrlA mutations. Addition of purified SecYEG restores the signal peptide specificity and increases protein translocation and ion channel activities. These data show that SecA can promote protein translocation and ion channel activities both when it is bound to lipids at low affinity sites and when it is bound to SecYEG with high affinity. The latter of the two interactions confers high efficiency and specificity.

Bowl-shaped oligomeric structures on membranes as DegP's new functional forms in protein quality control.

In the periplasm of Escherichia coli, DegP (also known as HtrA), which has both chaperone-like and proteolytic activities, prevents the accumulation of toxic misfolded and unfolded polypeptides. In solution, upon binding to denatured proteins, DegP forms large cage-like structures. Here, we show that DegP forms a range of bowl-shaped structures, independent of substrate proteins, each with a 4-, 5-, or 6-fold symmetry and all with a DegP trimer as the structural unit, on lipid membranes. These membrane-bound DegP assemblies have the capacity to recruit and process substrates in the bowl chamber, and they exhibit higher proteolytic and lower chaperone-like activities than DegP in solution. Our findings imply that DegP might regulate its dual roles during protein quality control, depending on its assembly state in the narrow bacterial envelope.