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Acta Cryst. (2014). A70, C243
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The tripartite motif (TRIM) proteins are a large family of >100 members, several of which have important roles in antiviral immunity and innate immune signaling. TRIM5α associates with incoming HIV-1 capsids, interfering with controlled disassembly and targeting them for degradation by the proteasome. TRIM21 is a cytosolic antibody receptor, which also targets incoming viral capsids for proteasomal degradation. TRIM25 is also involved in innate immunity, being essential for the ubiquitination of RIG-I. Recent positive selection analysis has predicted another 10 TRIM proteins with antiviral activity. Despite the fact that TRIM5α, 21 and 25 play key roles in antiviral protection, their mechanism of action is incompletely understood. All three proteins share a similar domain architecture, comprising a RING, B Box, coiled coil and PRYSPRY domains. The RING domains are responsible for ubiquitin ligase activity, while the PRYSPRY domains determine target specificity. We have used a combination of crystallography and SAXS to generate the first complete model for a TRIM protein structure. Crystallographic studies of TRIM25 reveal a central elongated coiled-coil domain with an unusual right-handed twist. The dimer formed by the coiled-coil is antiparallel but is followed by additional helices that reverse the direction of the protein chain. This structure suggests that the N-terminal domains of each monomer are separated but the C terminal domains are maintained in proximity. Multi-angle light scattering (MALS), isothermal titration calorimetry (ITC) and SAXS analysis confirms that this dimer structure is present in solution. Furthermore, scattering studies on the tripartite motif of TRIM21, comprising RING, B Box and coiled-coil, demonstrate that the first two domains of each monomer are held 150-200 Å apart. Finally, SAXS measurement of a complex between intact TRIM21 and its ligand, IgG Fc, provides the first empirical structure of a complete TRIM protein.

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Acta Cryst. (2014). A70, C404
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Small-angle X-ray scattering (SAXS) experiences a renaissance in the studies of macromolecular solutions allowing one to study the structure of native particles and complexes and to rapidly analyze structural changes in response to variations in external conditions. New high brilliance sources and novel data analysis methods significantly enhanced resolution and reliability of structural models provided by the technique (Graewert & Svergun, 2013). Automation of the experiment, data processing and interpretation make solution SAXS a streamline tool for large scale structural studies in molecular biology. The recent developments will be presented including robotic sample changers, pipelines for data processing, computation of structural parameters and ab initio models, classification of the folding states of macromolecules. A prototype of an expert systems allowing for automated generation and assessment of structural models will be considered. A synergistic use of SAXS with the high resolution methods like crystallography and NMR, but also with complementary biophysical and biochemical techniques will be discussed. The problems of validation of SAXS-generated models, and the use of data quality assessment tools for the deposition of the models and experimental data will be discussed. Further perspectives of the hybrid applications of SAXS with other techniques in structural biology will be outlined.

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Acta Cryst. (2014). A70, C405
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Hybrid approaches in structural biology have had a tremendous impact on our ability to tackle complex biological problems including large and flexible protein-protein assemblies. We have been employing creative combinations of X-ray crystallography, Small-angle X-ray Scattering (SAXS) and electron microscopy in conjunction with molecular interaction studies and cellular interrogation of the systems under study to elucidate the structural and mechanistic principles underlying diverse cytokine-receptor assemblies. Our studies have revealed the unexpected structural diversity of such assemblies, and have established that structural plasticity and molecular cooperativity both at the level of the cytokines and the receptors play critical roles in the assembly of signaling complexes. My presentation will provide a coherent overview of how we have tackled cytokine-receptor signalling complexes in a hypothesis-driven manner with the help of hybrid approaches in structural biology.

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Acta Cryst. (2014). A70, C425
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Proteins containing significant disordered regions are difficult for crystallization. Consequently, individual structured domains or subunits are often crystallized to determine their high resolution structures. These structures, not containing the disordered parts, are available from the protein databases. Small-Angle X-ray Scattering (SAXS) in solution is a complementary structural technique offering the possibility to characterize full length proteins including inter-domain disordered regions. Modern approaches for the analysis of the SAXS data from flexible systems utilize the representation of the system as ensembles of co-existing structures (e.g. Ensemble Optimization Method (EOM), Bernado et al. 2007). Here, the high resolution models of the domains are employed as rigid bodies together with the flexible parts represented by chains of dummy residues in order to construct models covering the configurational space of the full length proteins. Until now, the use of the ensemble approach was mostly limited to relatively simple cases like single chain proteins. We present improvements for the SAXS modeling including the possibilities to characterize flexible particles for complicated scenarios, in particular, for the cases when disorder comes in combination with point group symmetry. A new strategy for missing sections generation allows a rapid and accurate construction of the full length protein. Further, two metrics, Rflex and Rrat, are introduced for a quantitative assessment of the EOM results. Rflex is used as a measure of flexibility - based on the concept of entropy (information communication) - whereas Rrat is employed as a control parameter to detect potential artifacts. These developments implemented in the new version EOM 2.0 further promote the hybrid approach synergistically employing SAXS and MX in the analysis of complicated flexible systems. The capacity of the enhanced method will be illustrated by practical examples.

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Acta Cryst. (2014). A70, C431
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α-Actinin is the major component of the Z-disk, where it cross-links actin filaments from adjacent sarcomeres. It is an antiparallel dimer of 200 kDa, containing in each subunit an N-terminal actin binding domain (ABD), a central rod domain assembled from spectrin-like repeats that mediate the antiparallel assembly, and a C-terminal calmodulin-like (CaM-like) domain with 4 EF-hand motifs. Additionally to actin filaments, α-actinin binds multiple other cytoskeletal and signalling proteins. In striated muscle, the tightly defined numbers of α-actinin crosslinks between the antiparallel actin filaments at the Z-disk are organised by specific binding sites on the giant molecular blueprint of the sarcomere, titin. These titin Z-repeats contain a short, hydrophobic, α-actinin binding motif. To achieve ordered cytoskeletal assemblies, the binding properties of α-actinin must be tightly spatiotemporally regulated, in muscle α-actinin its actin and titin binding properties are regulated by phosphoinositide. Biochemical analyses led to propose previously that the α-actinin - titin interaction is regulated by an intramolecular mechanism, where the short sequence between the ABD and the rod interacts with the CaM-like domain in a pseudoligand complex, acting effectively as an intramolecular autoinhibitor. Here, we present the first complete crystallographic structure of sarcomeric human α-actinin complemented by small angle X-ray scattering data, electron-electron paramagnetic resonance, biochemical and in vivo cell biophysics studies of structure-informed mutants, which give insight into its molecular assembly and Z-disk architecture as well as into the mechanism of α-actinin function and regulation.
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