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γ-Secretase is an intramembrane protease complex that mediates the Notch signaling pathway and the production of amyloid β-proteins. As such, this enzyme has emerged as an important target for development of novel therapeutics for Alzheimer disease and cancer. Great progress has been made in the identification and characterization of the membrane complex and its biological functions. One major challenge now is to illuminate the structure of this fascinating and important protease at atomic resolution. Here, we review recent progress on biochemical and biophysical probing of the structure of the four-component complex and discuss obstacles and potential pathways toward elucidating its detailed structure. [Copyright &y& Elsevier]

We have obtained a 3D reconstruction of intact microtubules, using cryoelectron microscopy and image processing, at a resolution of about 8 A˚, sufficient to resolve much of the secondary structure. The level of detail in the map allows docking of the tubulin structure previously determined by electron crystallography, with very strong constraints, providing several important insights not previously available through docking tubulin into lower-resolution maps. This work provides an improved picture of the interactions between adjacent protofilaments, which are responsible for microtubule stability, and also suggests that some structural features are different in microtubules from those in the zinc sheets with which the tubulin structure was determined. [Copyright &y& Elsevier]

The human UBR5 is a single polypeptide chain homology to E6AP C terminus (HECT)-type E3 ubiquitin ligase essential for embryonic development in mammals. Dysregulated UBR5 functions like an oncoprotein to promote cancer growth and metastasis. Here, we report that UBR5 assembles into a dimer and a tetramer. Our cryoelectron microscopy (cryo-EM) structures reveal that two crescent-shaped UBR5 monomers assemble head to tail to form the dimer, and two dimers bind face to face to form the cage-like tetramer with all four catalytic HECT domains facing the central cavity. Importantly, the N-terminal region of one subunit and the HECT of the other form an "intermolecular jaw" in the dimer. We show the jaw-lining residues are important for function, suggesting that the intermolecular jaw functions to recruit ubiquitin-loaded E2 to UBR5. Further work is needed to understand how oligomerization regulates UBR5 ligase activity. This work provides a framework for structure-based anticancer drug development and contributes to a growing appreciation of E3 ligase diversity. [Display omitted] • The human UBR5 assembles into a dimer and a tetramer in solution • Cryo-EM structures of the UBR5 dimer and tetramer at 2.8 and 3.5 Å, respectively • Two UBR5 monomers assemble head to tail to form the dimer • Two UBR5 dimers assemble face to face to form the cage-like UBR5 tetramer Human UBR5 is an E3 ubiquitin ligase. Wang et al. discovered that UBR5 exists as dimers and tetramers in solution. They determined the dimer and tetramer structures to high resolution. The structures lay the foundation for elucidating the mechanism of UBR5 activity and developing anticancer drugs targeting the enzyme. [ABSTRACT FROM AUTHOR]

Summary: The eukaryotic origin recognition complex (ORC) interacts with and remodels origins of DNA replication prior to initiation in S phase. Here, we report a single-particle cryo-EM-derived structure of the supramolecular assembly comprising Saccharomyces cerevisiae ORC, the replication initiation factor Cdc6, and double-stranded ARS1 origin DNA in the presence of ATPγS. The six subunits of ORC are arranged as Orc1:Orc4:Orc5:Orc2:Orc3, with Orc6 binding to Orc2. Cdc6 binding changes the conformation of ORC, in particular reorienting the Orc1 N-terminal BAH domain. Segmentation of the 3D map of ORC-Cdc6 on DNA and docking with the crystal structure of the homologous archaeal Orc1/Cdc6 protein suggest an origin DNA binding model in which the DNA tracks along the interior surface of the crescent-like ORC. Thus, ORC bends and wraps the DNA. This model is consistent with the observation that binding of a single Cdc6 extends the ORC footprint on origin DNA from both ends. [ABSTRACT FROM AUTHOR]

Summary: Proteasome-mediated protein turnover in all domains of life is an energy-dependent process that requires ATPase activity. Mycobacterium tuberculosis (Mtb) was recently shown to possess a ubiquitin-like proteasome pathway that plays an essential role in Mtb resistance to killing by products of host macrophages. Here we report our structural and biochemical investigation of Mpa, the presumptive Mtb proteasomal ATPase. We demonstrate that Mpa binds to the Mtb proteasome in the presence of ATPγS, providing the physical evidence that Mpa is the proteasomal ATPase. X-ray crystallographic determination of the conserved interdomain showed a five stranded double β barrel structure containing a Greek key motif. Structure and mutational analysis indicate a major role of the interdomain for Mpa hexamerization. Our mutational and functional studies further suggest that the central channel in the Mpa hexamer is involved in protein substrate translocation and degradation. These studies provide insights into how a bacterial proteasomal ATPase interacts with and facilitates protein degradation by the proteasome. [Copyright &y& Elsevier]

Summary: Oligosaccharyl transferase (OT) catalyzes the transfer of a lipid-linked oligosaccharide to the nascent polypeptide emerging from the translocon. Currently, there is no structural information on the membrane-embedded OT complex, which consists of eight different polypeptide chains. We report a 12 Å resolution cryo-electron microscopy structure of OT from yeast. We mapped the locations of four essential OT subunits through a maltose-binding protein fusion strategy. OT was found to have a large domain in the lumenal side of endoplasmic reticulum where the catalysis occurs. The lumenal domain mainly comprises the catalytic Stt3p, the donor substrate-recognizing Wbp1p, and the acceptor substrate-recognizing Ost1p. A prominent groove was observed between these subunits, and we propose that the nascent polypeptide from the translocon threads through this groove while being scanned by the Ost1p subunit for the presence of the glycosylation sequon. [Copyright &y& Elsevier]

Packaging of the RNA molecule in viruses is important for the preservation and expression of viral genomic information. The vesicular stomatitis virus (VSV) nucleoproteins are kept associated with its negative-strand RNA during the mRNA synthesis and replication, in contrast to the tobacco mosaic virus whose nucleoproteins are released from RNA. It has been a puzzle how the VSV RNA is packaged to meet the contradicting requirements of protection and the accessibility to the polymerase. We report an 18 A˚ resolution structure of the recombinant nucleoprotein-RNA complex determined by single-particle electron microscopy. In the 3D density map, a ring of density is resolved on the inner surface and the density is proposed to be the RNA. The RNA is located on the inner surface of the decameric complex near the top end. This is dramatically different from the RNA packaging in TMV, but consistent with previously published biochemical findings. [Copyright &y& Elsevier]