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The dihydrolipoyl acyl-transferase (E2) enzyme forms the structural and catalytic core of the tripartite 2-oxoacid dehydrogenase multienzyme complexes of the central metabolic pathways. Although this family of multienzyme complexes shares a common architecture, their E2 cores form homo-trimers that, depending on the source, further associate into either octahedral (24-mer) or icosahedral (60-mer) assemblies, as predicted by the principles of quasi-equivalence. In the crystal structure of the E2 core from Thermoplasma acidophilum, a thermophilic archaeon, the homo-trimers assemble into a unique 42-mer oblate spheroid. Analytical equilibrium centrifugation and small-angle X-ray scattering analyses confirm that this catalytically active 1.08 MDa assembly exists as a single species in solution, forming a hollow spheroid with a maximum diameter of 220 Å. In this paper we show that a monodisperse macromolecular assembly, built from identical subunits in non-identical environments, forms an irregular protein shell via non-equivalent interactions. This unusually irregular protein shell, combining cubic and dodecahedral geometrical elements, expands on the concept of quasi-equivalence as a basis for understanding macromolecular assemblies by showing that cubic point group symmetry is not a physical requirement in multienzyme assembly. These results extend our basic knowledge of protein assembly and greatly expand the number of possibilities to manipulate self-assembling biological complexes to be utilized in innovative nanotechnology applications.

International audience; The three-dimensional structure of the hexameric (alpha beta)(6) 1.2-MDa complex formed by glutamate synthase has been determined at subnanometric resolution by combining cryoelectron microscopy, small angle x-ray scattering, and molecular modeling, providing for the first time a molecular model of this complex iron-sulfur flavoprotein. In the hexameric species, interprotomeric alpha-alpha and alpha-beta contacts are mediated by the C-terminal domain of the alpha subunit, which is based on a beta helical fold so far unique to glutamate synthases. The alpha beta protomer extracted from the hexameric model is fully consistent with it being the minimal catalytically active form of the enzyme. The structure clarifies the electron transfer pathway from the FAD cofactor on the beta subunit, to the FMN on the alpha subunit, through the low potential [4Fe-4S](1+/2+) centers on the beta subunit and the [3Fe-4S](0/1+) cluster on the alpha subunit. The(alpha beta)(6) hexamer exhibits a concentration-dependent equilibrium with alpha beta monomers and (alpha beta)(2) dimers, in solution, the hexamer being destabilized by high ionic strength and, to a lower extent, by the reaction product NADP(+). Hexamerization seems to decrease the catalytic efficiency of the alpha beta protomer only 3-fold by increasing the K-m values measured for L-Gln and 2-OG. However, it cannot be ruled out that the (alpha beta)(6) hexamer acts as a scaffold for the assembly of multienzymatic complexes of nitrogen metabolism or that it provides a means to regulate the activity of the enzyme through an as yet unknown ligand.

International audience The WeNMR (http://www.wenmr.eu) project is a European Union funded international effort to streamline and automate analysis of Nuclear Magnetic Resonance (NMR) and Small Angle X-Ray scattering (SAXS) imaging data for atomic and near-atomic resolution molecular structures. Conventional calculation of structure requires the use of various software packages, considerable user expertise and ample computational resources. To facilitate the use of NMR spectroscopy and SAXS in life sciences the WeNMR consortium has established standard computational workflows and services through easy-to-use web interfaces, while still retaining sufficient flexibility to handle more specific requests. Thus far, a number of programs often used in structural biology have been made available through application portals. The implementation of these services, in particular the distribution of calculations to a Grid computing infrastructure, involves a novel mechanism for submission and handling of jobs that is independent of the type of job being run. With over 450 registered users (September 2012), WeNMR is currently the largest Virtual Organization (VO) in life sciences. With its large and worldwide user community, WeNMR has become the first Virtual Research Community officially recognized by the European Grid Infrastructure (EGI).

The availability of new and fast tools in structure determination has led to a more than exponential growth of the number of structures solved per year. It is therefore increasingly essential to assess the accuracy of the new structures by reliable approaches able to assist validation. Here, we discuss a specific example in which the use of different complementary techniques, which include Bayesian methods and small angle scattering, resulted essential for validating the two currently available structures of the Josephin domain of ataxin-3, a protein involved in the ubiquitin/proteasome pathway and responsible for neurodegenerative spinocerebellar ataxia of type 3. Taken together, our results demonstrate that only one of the two structures is compatible with the experimental information. Based on the high precision of our refined structure, we show that Josephin contains an open cleft which could be directly implicated in the interaction with polyubiquitin chains and other partners.