Septins are GTP-binding proteins that assemble into homo- and hetero-oligomers and filaments. Although they have key roles in various cellular processes, little is known concerning the structure of septin subunits or the organization and polarity of septin complexes. Here we present the structures of the human SEPT2 G domain and the heterotrimeric human SEPT2-SEPT6-SEPT7 complex. The structures reveal a universal bipolar polymer building block, composed of an extended G domain, which forms oligomers and filaments by conserved interactions between adjacent nucleotide-binding sites and/or the amino- and carboxy-terminal extensions. Unexpectedly, X-ray crystallography and electron microscopy showed that the predicted coiled coils are not involved in or required for complex and/or filament formation. The asymmetrical heterotrimers associate head-to-head to form a hexameric unit that is nonpolarized along the filament axis but is rotationally asymmetrical. The architecture of septin filaments differs fundamentally from that of other cytoskeletal structures.

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We developed a method, named GraDeR, which substantially improves the preparation of membrane protein complexes for structure determination by single-particle cryo-electron microscopy (cryo-EM). In GraDeR, glycerol gradient centrifugation is used for the mild removal of free detergent monomers and micelles from lauryl maltose-neopentyl glycol detergent stabilized membrane complexes, resulting in monodisperse and stable complexes to which standard processes for water-soluble complexes can be applied. We demonstrate the applicability of the method on three different membrane complexes, including the mammalian FoF1 ATP synthase. For this highly dynamic and fragile rotary motor, we show that GraDeR allows visualizing the asymmetry of the F1 domain, which matches the ground state structure of the isolated domain. Therefore, the present cryo-EM structure of FoF1 ATP synthase provides direct structural evidence for Boyer's binding change mechanism in the context of the intact enzyme.

Background: Nucleoprotein (NP) is an essential component of the virus replication complex. Results:The crystal and quaternary structures of Lassa virus NP were determined. Conclusion: Lassa virus NP assembles into a symmetric trimer in solution.Significance: The trimeric complex may have a biological function in the virus life cycle, and its assembly could be a target for antivirals.

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Translation initiation in eukaryotes is a multistep process requiring the orchestrated interaction of several eukaryotic initiation factors (eIFs). The largest of these factors, eIF3, forms the scaffold for other initiation factors, promoting their binding to the 40S ribosomal subunit. Biochemical and structural studies on eIF3 need highly pure eIF3. However, natively purified eIF3 comprise complexes containing other proteins such as eIF5. Therefore we have established in vitro reconstitution protocols for Saccharomyces cerevisiae eIF3 using its five recombinantly expressed and purified subunits. This reconstituted eIF3 complex (eIF3 rec ) exhibits the same size and activity as the natively purified eIF3 (eIF3 nat ). The homogeneity and stoichiometry of eIF3 rec and eIF3 nat were confirmed by analytical size exclusion chromatography, mass spectrometry, and multi-angle light scattering, demonstrating the presence of one copy of each subunit in the eIF3 complex. The reconstituted and native eIF3 complexes were compared by single-particle electron microscopy showing a high degree of structural conservation. The interaction network between eIF3 proteins was studied by means of limited proteolysis, analytical size exclusion chromatography, in vitro binding assays, and isothermal titration calorimetry, unveiling distinct protein domains and subcomplexes that are critical for the integrity of the protein network in yeast eIF3. Taken together, the data presented here provide a novel procedure to obtain highly pure yeast eIF3, suitable for biochemical and structural analysis, in addition to a detailed picture of the network of protein interactions within this complex.

Most known methods for the determination of the structure of macromolecular complexes are limited or at least restricted at some point by their computational demands. Recent developments in information technology such as multicore, parallel and GPU processing can be used to overcome these limitations. In particular, graphics processing units (GPUs), which were originally developed for rendering real-time effects in computer games, are now ubiquitous and provide unprecedented computational power for scientific applications. Each parallel-processing paradigm alone can improve overall performance; the increased computational performance obtained by combining all paradigms, unleashing the full power of today's technology, makes certain applications feasible that were previously virtually impossible. In this article, state-of-the-art paradigms are introduced, the tools and infrastructure needed to apply these paradigms are presented and a state-of-the-art infrastructure and solution strategy for moving scientific applications to the next generation of computer hardware is outlined.

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