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Translocation of membrane proteins from the point of synthesis to their integration in the membrane is critical to the function of the cell. Tail-anchored (TA) proteins are an important class of membrane proteins with a single transmembrane domain (TMD) close to the carboxyl-terminus. They are defined topologically by having their amino-terminus in the cytosol and their carboxyl-terminus on the exterior side of the membrane. Since the TMD is sequestered by the ribosome during translation, co-translational translocation of TA proteins by the SRP-dependent pathway is not possible. The Guided Entry of Tail-anchored proteins (GET) pathway post-translationally targets TA proteins to the endoplasmic reticulum (ER) membrane. The conserved nucleotide hydrolase Get3 is the central protein in the pathway that specifically binds the TMD of TA proteins to chaperone them from a sorting complex of Get4, Get5, Sgt2 and other chaperones to an ER membrane receptor formed by Get1 and Get2. We have created a model for the mechanism of Get3 TA protein binding coupled to nucleotide state using X-ray crystallography, structural modeling and mutagenesis experiments. We then demonstrate expression, purification and crystallization of complexes of Get3 with TA proteins for structural studies. Finally, we present a crystal structure of a tetrameric archaeal Get3 homologue that forms a central hydrophobic chamber and is capable of binding TA proteins. Using small-angle X-ray scattering, the structure is comparable to a tetrameric fungal Get3 complex with TA protein, which is capable of TA protein membrane integration in vitro. This suggests a model in which a tetramer of Get3 binds TA proteins for delivery to the membrane.

Translocation of membrane proteins from the point of synthesis to their integration in the membrane is critical to the function of the cell. Tail-anchored (TA) proteins are an important class of membrane proteins with a single transmembrane domain (TMD) close to the carboxyl-terminus. They are defined topologically by having their amino-terminus in the cytosol and their carboxyl-terminus on the exterior side of the membrane. Since the TMD is sequestered by the ribosome during translation, co-translational translocation of TA proteins by the SRP-dependent pathway is not possible. The Guided Entry of Tail-anchored proteins (GET) pathway post-translationally targets TA proteins to the endoplasmic reticulum (ER) membrane. The conserved nucleotide hydrolase Get3 is the central protein in the pathway that specifically binds the TMD of TA proteins to chaperone them from a sorting complex of Get4, Get5, Sgt2 and other chaperones to an ER membrane receptor formed by Get1 and Get2. We have created a model for the mechanism of Get3 TA protein binding coupled to nucleotide state using X-ray crystallography, structural modeling and mutagenesis experiments. We then demonstrate expression, purification and crystallization of complexes of Get3 with TA proteins for structural studies. Finally, we present a crystal structure of a tetrameric archaeal Get3 homologue that forms a central hydrophobic chamber and is capable of binding TA proteins. Using small-angle X-ray scattering, the structure is comparable to a tetrameric fungal Get3 complex with TA protein, which is capable of TA protein membrane integration in vitro. This suggests a model in which a tetramer of Get3 binds TA proteins for delivery to the membrane.

Translocation of membrane proteins from the point of synthesis to their integration in the membrane is critical to the function of the cell. Tail-anchored (TA) proteins are an important class of membrane proteins with a single transmembrane domain (TMD) close to the carboxyl-terminus. They are defined topologically by having their amino-terminus in the cytosol and their carboxyl-terminus on the exterior side of the membrane. Since the TMD is sequestered by the ribosome during translation, co-translational translocation of TA proteins by the SRP-dependent pathway is not possible. The Guided Entry of Tail-anchored proteins (GET) pathway post-translationally targets TA proteins to the endoplasmic reticulum (ER) membrane. The conserved nucleotide hydrolase Get3 is the central protein in the pathway that specifically binds the TMD of TA proteins to chaperone them from a sorting complex of Get4, Get5, Sgt2 and other chaperones to an ER membrane receptor formed by Get1 and Get2. We have created a model for the mechanism of Get3 TA protein binding coupled to nucleotide state using X-ray crystallography, structural modeling and mutagenesis experiments. We then demonstrate expression, purification and crystallization of complexes of Get3 with TA proteins for structural studies. Finally, we present a crystal structure of a tetrameric archaeal Get3 homologue that forms a central hydrophobic chamber and is capable of binding TA proteins. Using small-angle X-ray scattering, the structure is comparable to a tetrameric fungal Get3 complex with TA protein, which is capable of TA protein membrane integration in vitro. This suggests a model in which a tetramer of Get3 binds TA proteins for delivery to the membrane.

The Influenza Research Database (IRD) is a U.S. National Institute of Allergy and Infectious Diseases (NIAID)-sponsored Bioinformatics Resource Center dedicated to providing bioinformatics support for influenza virus research. IRD facilitates the research and development of vaccines, diagnostics and therapeutics against influenza virus by providing a comprehensive collection of influenza-related data integrated from various sources, a growing suite of analysis and visualization tools for data mining and hypothesis generation, personal workbench spaces for data storage and sharing, and active user community support. Here, we describe the recent improvements in IRD including the use of cloud and high performance computing resources, analysis and visualization of user-provided sequence data with associated metadata, predictions of novel variant proteins, annotations of phenotype-associated sequence markers and their predicted phenotypic effects, hemagglutinin (HA) clade classifications, an automated tool for HA subtype numbering conversion, linkouts to disease event data and the addition of host factor and antiviral drug components. All data and tools are freely available without restriction from the IRD website at https://www.fludb.org.

Electron tomography is currently the highest resolution imaging modality available to study the 3D structures of pleomorphic macromolecular assemblies, viruses, organelles and cells. Unfortunately, the resolution is currently limited to 3–5 nm by several factors including the dose tolerance of biological specimens and the inaccessibility of certain tilt angles. Here we report the first experimental demonstration of equally-sloped tomography (EST) to alleviate these problems. As a proof of principle, we applied EST to reconstructing frozen-hydrated keyhole limpet hemocyanin molecules from a tilt-series taken with constant slope increments. In comparison with weighted back-projection (WBP), the algebraic reconstruction technique (ART) and the simultaneous algebraic reconstruction technique (SART), EST reconstructions exhibited higher contrast, less peripheral noise, more easily detectable molecular boundaries and reduced missing wedge effects. More importantly, EST reconstructions including only two-thirds the original images appeared to have the same resolution as full WBP reconstructions, suggesting that EST can either reduce the dose required to reach a given resolution or allow higher resolutions to be achieved with a given dose. EST was also applied to reconstructing a frozen-hydrated bacterial cell from a tilt-series taken with constant angular increments. The results confirmed similar benefits when standard tilts are utilized.

Electron tomography is currently the highest resolution imaging modality available to study the 3D structures of pleomorphic macromolecular assemblies, viruses, organelles and cells. Unfortunately, the resolution is currently limited to 3–5 nm by several factors including the dose tolerance of biological specimens and the inaccessibility of certain tilt angles. Here we report the first experimental demonstration of equally-sloped tomography (EST) to alleviate these problems. As a proof of principle, we applied EST to reconstructing frozen-hydrated keyhole limpet hemocyanin molecules from a tilt-series taken with constant slope increments. In comparison with weighted back-projection (WBP), the algebraic reconstruction technique (ART) and the simultaneous algebraic reconstruction technique (SART), EST reconstructions exhibited higher contrast, less peripheral noise, more easily detectable molecular boundaries and reduced missing wedge effects. More importantly, EST reconstructions including only two-thirds the original images appeared to have the same resolution as full WBP reconstructions, suggesting that EST can either reduce the dose required to reach a given resolution or allow higher resolutions to be achieved with a given dose. EST was also applied to reconstructing a frozen-hydrated bacterial cell from a tilt-series taken with constant angular increments. The results confirmed similar benefits when standard tilts are utilized.