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With the rapid improvement of cryo-electron microscopy (cryo-EM) resolution, new computational tools are needed to assist and improve upon atomic model building and refinement options. This communication demonstrates that microscopists can now collaborate with the players of the computer game Foldit to generate high-quality de novo structural models. This development could greatly speed the generation of excellent cryo-EM structures when used in addition to current methods. This Community Page article demonstrates that microscopists can now collaborate with the players of the computer game Foldit to generate thorough high-quality de novo structural models; this development could greatly speed the generation of excellent Cryo-EM structures when used as a complement to current methods. [ABSTRACT FROM AUTHOR]

Measles is a highly contagious human disease. We used cryo--electron microscopy and single particle--based helical image analysis to determine the structure of the helical nucleocapsid formed by the folded domain of the measles virus nucleoprotein encapsidating an RNA at a resolution of 4.3 angstroms. The resulting pseudoatomic model of the measles virus nucleocapsid offers important insights into the mechanism of the helical polymerization of nucleocapsids of negative-strand RNA viruses, in particular via the exchange subdomains of the nucleoprotein. The structure reveals the mode of the nucleoprotein-RNA interaction and explains why each nucleoprotein ofmeasles virus binds six nucleotides,whereas the respiratory syncytial virus nucleoprotein binds seven. It provides a rational basis for further analysis of measles virus replication and transcription, and reveals potential targets for drug design. [ABSTRACT FROM AUTHOR]

Summary: Hexameric DnaB helicases are often loaded at DNA replication forks by interacting with the initiator protein DnaA and/or a helicase loader (DnaC in Escherichia coli). These loaders are not universally required, and DnaB from Helicobacter pylori was found to bypass DnaC when expressed in E. coli cells. The crystal structure of Helicobacter pylori DnaB C-terminal domain (HpDnaB-CTD) reveals a large two-helix insertion (named HPI) in the ATPase domain that protrudes away from the RecA fold. Biophysical characterization and electron microscopy (EM) analysis of the full-length protein show that HpDnaB forms head-to-head double hexamers remarkably similar to helicases found in some eukaryotes, archaea, and viruses. The docking of the HpDnaB-CTD structure into EM reconstruction of HpDnaB provides a model that shows how hexamerization of the CTD is facilitated by HPI-HPI interactions. The HpDnaB double-hexamer architecture supports an alternative strategy to load bacterial helicases onto forks in the absence of helicase loaders. [ABSTRACT FROM AUTHOR]

Abstract: In order to preserve their genome integrity, organisms have developed elaborate tactics for genome protection and repair. The Deinococcus radiodurans bacteria famous for their extraordinary tolerance toward high doses of radiations or long period of desiccation, possess some specific genes with unknown function which are related to their survival in such extreme conditions. Among them, ddrA is an orphan gene specific of Deinococcus genomes. DdrA, the product of this gene was suggested to be a component of the DNA end protection system. Here we provide a three-dimensional reconstruction of the Deinococcus deserti DdrA(1–160) by electron microscopy. Although not functional in vivo, this truncated protein keeps its DNA binding ability at the wild-type level. DdrA(1–160) has a complex three-dimensional structure based on a heptameric ring that can self-associate to form a larger molecular weight assembly. We suggest that the complex architecture of DdrA plays a role in the substrate specificity and favors an efficient DNA repair. [Copyright &y& Elsevier]

A major effort has been made by the structural biology community to develop user-friendly software for the use of biologists. However, structural projects become more and more challenging and their solution often relies on a combination of information from various sources. Here, it is described how X-ray data, normal-mode analysis (NMA) and electron-microscopy (EM) data can be successfully combined in order to obtain a molecular-replacement (MR) solution for crystal structures containing multimeric molecules. NMA is used to simulate computationally the inherent internal flexibility of the monomer and thus enhance, together with the crystal noncrystallographic symmetry (NCS), the MR capabilities. NCS is also used to obtain a reliable EM reconstruction, which is then employed as a filter to construct oligomers starting from monomers. The feasibility of the direct use of EM reconstructions as a template for MR when the X-­ray and EM data resolutions overlap is also discussed. [ABSTRACT FROM AUTHOR]

Summary Roundabout (Robo) receptors provide an essential repulsive cue in neuronal development following Slit ligand binding. This important signaling pathway can also be hijacked in numerous cancers, making Slit-Robo an attractive therapeutic target. However, little is known about how Slit binding mediates Robo activation. Here we present the crystal structure of Robo1 Ig1-4 and Robo1 Ig5, together with a negative stain electron microscopy reconstruction of the Robo1 ectodomain. These results show how the Robo1 ectodomain is arranged as compact dimers, mainly mediated by the central Ig domains, which can further interact in a “back-to-back” fashion to generate a tetrameric assembly. We also observed no change in Robo1 oligomerization upon interaction with the dimeric Slit2-N ligand using fluorescent imaging. Taken together with previous studies we propose that Slit2-N binding results in a conformational change of Robo1 to trigger cell signaling. [ABSTRACT FROM AUTHOR]

Abstract: Helical reconstruction from electron cryomicrographs has become a routine technique for macromolecular structure determination of helical assemblies since the first days of Fourier-based three-dimensional image reconstruction. In the past decade, the single-particle technique has had an important impact on the advancement of helical reconstruction. Here, we present the software package SPRING that combines Fourier based symmetry analysis and real-space helical processing into a single workflow. One of the most time-consuming steps in helical reconstruction is the determination of the initial symmetry parameters. First, we propose a class-based helical reconstruction approach that enables the simultaneous exploration and evaluation of many symmetry combinations at low resolution. Second, multiple symmetry solutions can be further assessed and refined by single-particle based helical reconstruction using the correlation of simulated and experimental power spectra. Finally, the 3D structure can be determined to high resolution. In order to validate the procedure, we use the reference specimen Tobacco Mosaic Virus (TMV). After refinement of the helical symmetry, a total of 50,000 asymmetric units from two micrographs are sufficient to reconstruct a subnanometer 3D structure of TMV at 6.4Å resolution. Furthermore, we introduce the individual programs of the software and discuss enhancements of the helical reconstruction workflow. Thanks to its user-friendly interface and documentation, SPRING can be utilized by the novice as well as the expert user. In addition to the study of well-ordered helical structures, the development of a streamlined workflow for single-particle based helical reconstruction opens new possibilities to analyze specimens that are heterogeneous in symmetries. [Copyright &y& Elsevier]

Recombinant measles virus nucleoprotein-RNA (N-RNA) helices were analyzed by negative-stain electron microscopy. Three-dimensional reconstructions of trypsin-digested and intact nucleocapsids coupled to the docking of the atomic structure of the respiratory syncytial virus (RSV) N-RNA subunit into the electron microscopy density map support a model that places the RNA at the exterior of the helix and the disordered C-terminal domain toward the helix interior, and they suggest the position of the six nucleotides with respect to the measles N protomer. [ABSTRACT FROM AUTHOR]