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.

Mechanisms of ribosome stalling by SecM at multiple elongation steps.

Regulation of translating ribosomes is a major component of gene expression control network. In Escherichia coli, ribosome stalling by the C-terminal arrest sequence of SecM regulates the SecA-dependent secretion pathway. Previous studies reported many residues of SecM peptide and ribosome exit tunnel are critical for stalling. However, the underlying molecular mechanism is still not clear at the atomic level. Here, we present two cryo-EM structures of the SecM-stalled ribosomes at 3.3-3.7 Å resolution, which reveal two different stalling mechanisms at distinct elongation steps of the translation cycle: one is due to the inactivation of ribosomal peptidyl-transferase center which inhibits peptide bond formation with the incoming prolyl-tRNA; the other is the prolonged residence of the peptidyl-RNA at the hybrid A/P site which inhibits the full-scale tRNA translocation. These results demonstrate an elegant control of translation cycle by regulatory peptides through a continuous, dynamic reshaping of the functional center of the ribosome.

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.

Structural characterization of full-length NSF and 20S particles.

The 20S particle, which is composed of the N-ethylmaleimide-sensitive factor (NSF), soluble NSF attachment proteins (SNAPs) and the SNAP receptor (SNARE) complex, has an essential role in intracellular vesicle fusion events. Using single-particle cryo-EM and negative stain EM, we reconstructed four related three-dimensional structures: Chinese hamster NSF hexamer in the ATPγS, ADP-AlFx and ADP states, and the 20S particle. These structures reveal a parallel arrangement between the D1 and D2 domains of the hexameric NSF and characterize the nucleotide-dependent conformational changes in NSF. The structure of the 20S particle shows that it holds the SNARE complex at two interaction interfaces around the C terminus and N-terminal half of the SNARE complex, respectively. These findings provide insight into the molecular mechanism underlying disassembly of the SNARE complex by NSF.

Characterization of the structure and function of Escherichia coli DegQ as a representative of the DegQ-like proteases of bacterial HtrA family proteins.

HtrA family proteins play a central role in protein quality control in the bacterial periplasmic space. DegQ-like proteases, a group of bacterial HtrA proteins, are characterized by a short LA loop as compared with DegP-like proteases, and are found in many bacterial species. As a representative of the DegQ-like proteases, we report that Escherichia coli DegQ exists in vivo primarily as a trimer (substrate-free) or dodecamer (substrate-containing). Biochemical analysis of DegQ dodecamers revealed that the major copurified protein substrate is OmpA. Importantly, wild-type DegQ exhibited a much lower proteolytic activity, and thus higher chaperone-like activity, than DegP. Furthermore, using cryo-electron microscopy we determined high-resolution structures of DegQ 12- and 24-mers in the presence of substrate, thus revealing the structural mechanism by which DegQ moderates its proteolytic activity.

Dimeric SecA couples the preprotein translocation in an asymmetric manner.

The Sec translocase mediates the post-translational translocation of a number of preproteins through the inner membrane in bacteria. In the initiatory translocation step, SecB targets the preprotein to the translocase by specific interaction with its receptor SecA. The latter is the ATPase of Sec translocase which mediates the post-translational translocation of preprotein through the protein-conducting channel SecYEG in the bacterial inner membrane. We examined the structures of Escherichia coli Sec intermediates in solution as visualized by negatively stained electron microscopy in order to probe the oligomeric states of SecA during this process. The symmetric interaction pattern between the SecA dimer and SecB becomes asymmetric in the presence of proOmpA, and one of the SecA protomers predominantly binds to SecB/proOmpA. Our results suggest that during preprotein translocation, the two SecA protomers are different in structure and may play different roles.

Electron microscopic visualization of asymmetric precursor translocation intermediates: SecA functions as a dimer.

SecA, the ATPase of Sec translocase, mediates the post-translational translocation of preprotein through the protein-conducting channel SecYEG in the bacterial inner membrane. Here we report the structures of Escherichia coli Sec intermediates during preprotein translocation as visualized by electron microscopy to probe the oligomeric states of SecA during this process. We found that the translocase holoenzyme is symmetrically assembled by SecA and SecYEG on proteoliposomes, whereas the translocation intermediate 31 (I(31)) becomes asymmetric because of the presence of preprotein. Moreover, SecA is a dimer in these two translocation complexes. This work also shows surface topological changes in the components of translocation intermediates by immunogold labeling. The channel entry for preprotein translocation was found at the center of the I(31) structures. Our results indicate that the presence of preprotein introduces asymmetry into translocation intermediates, while SecA remains dimeric during the translocation process.

The structure of SecB/OmpA as visualized by electron microscopy: The mature region of the precursor protein binds asymmetrically to SecB.

SecB, a molecular chaperone in Escherichia coli, binds a subset of precursor proteins that are exported across the plasma membrane via the Sec pathway. Previous studies showed that SecB bound directly to the mature region rather than to the signal sequence of the precursor protein. To determine the binding pattern of SecB and the mature region of the preprotein, here, we visualized the structure of the SecB/OmpA complex by electron microscopy. This complex is composed by two parts: the main density represents one SecB tetramer and the unfolded part of OmpA wrapping round it; the elongated smaller density represents the rest of OmpA. Each SecB protomer makes a different contribution to the binding of SecB with OmpA. The binding pattern between SecB tetramer and OmpA is asymmetric.

Full-length Escherichia coli SecA dimerizes in a closed conformation in solution as determined by cryo-electron microscopy.

SecA is an obligatory component of the Escherichia coli general secretion pathway. However, the oligomeric structure of SecA and SecA conformational changes during translocation processes are still unclear. Here we obtained the three-dimensional structure of E. coli wild-type full-length SecA in solution by single particle cryo-electron microscopy and determined its oligomeric organization. In this structure, SecA occurs as a dimer in which the two protomers are arranged in an antiparallel mode, with a novel electrostatic interface, and both protomers are in closed conformation. The system developed here may provide a promising technique for studying dynamic structural changes in SecA.