Publication date: June 2013
Source:Journal of Structural Biology, Volume 182, Issue 3

Publication date: June 2013
Source:Journal of Structural Biology, Volume 182, Issue 3

Publication date: June 2013
Source:Journal of Structural Biology, Volume 182, Issue 3

Author(s): Peter D. Simone , Lucas R. Struble , Admir Kellezi , Carrie A. Brown , Corinn E. Grabow , Irine Khutsishvili , Luis A. Marky , Youri I. Pavlov , Gloria E.O. Borgstahl

Inosine triphosphate pyrophosphatase (ITPA), a key enzyme involved in maintaining the purity of cellular nucleoside triphosphate pools, specifically recognizes inosine triphosphate and xanthosine triphosphate (including the deoxyribose forms) and detoxifies them by catalyzing the hydrolysis of a phosphoanhydride bond, releasing pyrophosphate. This prevents their inappropriate use as substrates in enzymatic reactions utilizing (d)ATP or (d)GTP. A human genetic polymorphism leads to the substitution of Thr for Pro32 (P32T) and causes ITPA deficiency in erythrocytes, with heterozygotes having on average 22.5% residual activity, and homozygotes having undetectable activity. This polymorphism has been implicated in modulating patients’ response to mercaptopurines and ribavirin. Human fibroblasts containing this variant have elevated genomic instability upon treatment with base analogs. We find that the wild-type and P32T forms are dimeric in solution and in the crystal structure. This abolishes the previous speculation that the P32T change disrupts dimerization as a mechanism of inactivation. The only difference in structure from the wild-type protein is that the area surrounding Thr32 is disrupted. Phe31 is flipped from the hydrophobic core out into the solvent, leaving a hole in the hydrophobic core of the protein which likely accounts for the reduced thermal stability of P32T ITPA and ultimately leads to its susceptibility to degradation in human cells. Circular dichroism and thermal denaturation studies confirm these structural results. We propose that the dimer of P32T variant subunit with wild-type subunit is degraded in cells similarly to the P32T homodimer explaining the level of loss of ITPA activity in heterozygotes.

Publication date: June 2013
Source:Journal of Structural Biology, Volume 182, Issue 3

Author(s): Fotis A. Baltoumas , Margarita C. Theodoropoulou , Stavros J. Hamodrakas

G-protein coupled receptors (GPCRs) are one of the largest families of membrane receptors in eukaryotes. Heterotrimeric G-proteins, composed of α, β and γ subunits, are important molecular switches in the mediation of GPCR signaling. Receptor stimulation after the binding of a suitable ligand leads to G-protein heterotrimer activation and dissociation into the Gα subunit and Gβγ heterodimer. These subunits then interact with a large number of effectors, leading to several cell responses. We studied the interactions between Gα subunits and their binding partners, using information from structural, mutagenesis and Bioinformatics studies, and conducted a series of comparisons of sequence, structure, electrostatic properties and intermolecular energies among different Gα families and subfamilies. We identified a number of Gα surfaces that may, in several occasions, participate in interactions with receptors as well as effectors. The study of Gα interacting surfaces in terms of sequence, structure and electrostatic potential reveals features that may account for the Gα subunit’s behavior towards its interacting partners. The electrostatic properties of the Gα subunits, which in some cases differ greatly not only between families but also between subfamilies, as well as the G-protein interacting surfaces of effectors and regulators of G-protein signaling (RGS) suggest that electrostatic complementarity may be an important factor in G-protein interactions. Energy calculations also support this notion. This information may be useful in future studies of G-protein interactions with GPCRs and effectors.

Publication date: June 2013
Source:Journal of Structural Biology, Volume 182, Issue 3

Author(s): Lisbeth G. Thygesen , Notburga Gierlinger

Polarised Raman micrsospectroscopy was employed to study the molecular structure within dislocations (slip planes) in the cell walls of Hemp fibre cells (Cannabis sativa (L.)). It was found that the cellulose microfibrils within dislocations have a different orientation than in the surrounding cell wall, and that the cellulose in the transition zones between a large dislocation and the surrounding wall may have yet another orientation. Furthermore, cellulose orientation seemed to be less uniform within dislocations than in the surrounding cell wall.

Publication date: June 2013
Source:Journal of Structural Biology, Volume 182, Issue 3

Author(s): Alessandra Patera , Dominique Derome , Michele Griffa , Jan Carmeliet

The swelling and shrinkage of four Picea abies (L. Karst) wood tissue homogeneous samples, of porosity varying between 45% and 78%, is documented with high-resolution synchrotron radiation phase-contrast X-ray tomographic microscopy. We report measurements of the reversible moisture-induced orthotropic swelling/shrinkage strains. Hysteresis is observed when the swelling/shrinkage strain is considered as a function of relative humidity, except for the very high porosity sample. Hysteresis is no longer present when swelling/shrinkage strains are considered versus moisture content, indicating that wood deforms to the same extent whether an amount of moisture is desorbed or adsorbed. Furthermore, swelling anisotropy, in the tangential and radial directions, is found to increase with increasing porosity. The most homogeneous behaviour for a group of cells is found for 30–50 cells, smaller/larger groups having higher orders of variations.

Publication date: June 2013
Source:Journal of Structural Biology, Volume 182, Issue 3

Author(s): Yuxiang Chen , Stefan Pfeffer , Thomas Hrabe , Jan Michael Schuller , Friedrich Förster

In cryoelectron tomography alignment and averaging of subtomograms, each dnepicting the same macromolecule, improves the resolution compared to the individual subtomogram. Major challenges of subtomogram alignment are noise enhancement due to overfitting, the bias of an initial reference in the iterative alignment process, and the computational cost of processing increasingly large amounts of data. Here, we propose an efficient and accurate alignment algorithm via a generalized convolution theorem, which allows computation of a constrained correlation function using spherical harmonics. This formulation increases computational speed of rotational matching dramatically compared to rotation search in Cartesian space without sacrificing accuracy in contrast to other spherical harmonic based approaches. Using this sampling method, a reference-free alignment procedure is proposed to tackle reference bias and overfitting, which also includes contrast transfer function correction by Wiener filtering. Application of the method to simulated data allowed us to obtain resolutions near the ground truth. For two experimental datasets, ribosomes from yeast lysate and purified 20S proteasomes, we achieved reconstructions of approximately 20Å and 16Å, respectively. The software is ready-to-use and made public to the community.

Publication date: June 2013
Source:Journal of Structural Biology, Volume 182, Issue 3

Author(s): Claudia Antoni , Laura Vera , Laurent Devel , Maria Pia Catalani , Bertrand Czarny , Evelyn Cassar-Lajeunesse , Elisa Nuti , Armando Rossello , Vincent Dive , Enrico Adriano Stura

Homodimerization is important in signal transduction and can play a crucial role in many other biological systems. To obtaining structural information for the design of molecules able to control the signalization pathways, the proteins involved will have to be crystallized in complex with ligands that induce dimerization. Bi-functional drugs have been generated by linking two ligands together chemically and the relative crystallizability of complexes with mono-functional and bi-functional ligands has been evaluated. There are problems associated with crystallization with such ligands, but overall, the advantages appear to be greater than the drawbacks. The study involves two matrix metalloproteinases, MMP-12 and MMP-9. Using flexible and rigid linkers we show that it is possible to control the crystal packing and that by changing the ligand-enzyme stoichiometric ratio, one can toggle between having one bi-functional ligand binding to two enzymes and having the same ligand bound to each enzyme. The nature of linker and its point of attachment on the ligand can be varied to aid crystallization, and such variations can also provide valuable structural information about the interactions made by the linker with the protein. We report here the crystallization and structure determination of seven ligand-dimerized complexes. These results suggest that the use of bi-functional drugs can be extended beyond the realm of protein dimerization to include all drug design projects.

Publication date: June 2013
Source:Journal of Structural Biology, Volume 182, Issue 3

Author(s): Ying Liu , Xing Meng , Zheng Liu

For biological samples showing a preferred orientation on the carbon support film of an electron microscope (EM) grid, accurate three-dimensional (3D) reconstructions by single-particle cryo-EM require data collection in which the specimen grids are tilted in the microscope, to obtain adequate numbers of particles that cover the high-degree angular distribution. However, image drift caused by the electron beam interacting with the cryo specimen becomes severe when grids are tilted to high angles (>30°). We produced deformed grids by applying a deliberate mechanical deformation to EM grids containing a thin carbon film supported by a thick holey carbon film. We applied cryo-EM using deformed grids to the isolated cardiac ryanodine receptor, an ion channel complex known to assume a preferred orientation on the carbon support film. These grids contained more particles having high Euler angle orientations without the need to tilt the specimen grids. Meanwhile, the drifting that was apparent in the images was reduced from that typical of images from tilted regular EM grids. This was achieved by imaging particles in holes close to the deformed areas, where carbon films were locally bent, offering planes of inclination with various angles. The deformed grids improve the efficiency and quality of data collection for single-particle cryo-EM of samples showing a limited range of orientations.

Publication date: June 2013
Source:Journal of Structural Biology, Volume 182, Issue 3

Author(s): Cyril Hamiaux , Lisa Basten , David R. Greenwood , Edward N. Baker , Richard D. Newcomb

Takeout proteins are found across a diverse range of insect species and are thought to be involved in important aspects of insect physiology and behavior. These proteins act as ligand carriers, but the nature of their endogenous ligands remains unknown. The crystal structure of Epiphyas postvittana Takeout 1 (EpTo1), the only structure for any Takeout protein to date, revealed an α/β-wrap fold with a purely hydrophobic internal cavity of tubular shape. When recombinantly expressed in Escherichia coli, we previously showed that a surrogate ubiquinone-8 ligand binds within the internal cavity of EpTo1 with excellent shape complementarity. We have now expressed EpTo1 in an insect cell expression system devoid of ubiquinone-8, and solved its crystal structure at 2.2Å resolution. Using combined information from crystallography and mass spectrometry, we identify a mixture of fatty acid moieties, mostly myristic and palmitic acid, bound inside the EpTo1 cavity, mimicking the structure of the longer ubiquinone-8 compound. No significant alteration of the internal cavity was observed regardless of the bound ligands, ubiquinone-8 or fatty acids, suggesting that the internal cavity of EpTo1 forms a rigid scaffold that imposes strict structural constraints for selectivity and specificity of ligand(s) in vivo.

Publication date: Available online 1 June 2013
Source:Journal of Structural Biology

Author(s): Jen-Wei Chang , Yi-Min Wu , Zi-Yun Chen , Shih-Hsin Huang , Chun-Hsiung Wang , Pei-lun Wu , Yi-ping Weng , Changjiang You , Jacob Piehler , Wei-hau Chang

TFIIF—a general transcription factor comprising two subunits Tfg1 and Tfg2, can tightly associate with RNA polymerase II (RNAPII) to regulate the mRNA synthesis in yeast. Herein, to localize the C-terminus of Tfg2 in the RNAPII-TFIIF architecture, we devise a two-stage hybrid approach using electron microscopy (EM) and Förster resonance energy transfer (FRET). In the first stage, we label the poly-histidine appended to the Tfg2 C-terminus with nickel-NTA nanogold and use a seven-step single particle image analysis protocol to limit the nanogold-accessible region next to the RNAPII clamp. In the second stage, for, Rpb2 and Rpb4 subunits of RNAPII proximal to the clamp are chosen for placing FRET satellites to enable nano-positioning (NP) analysis for improving the precision of localization. NP analysis shows the Tfg2 C-terminus is localized on the ridge of the clamp but may move to the top during transcription while the template DNA could be reciprocally perturbed by TFIIF.In summary, our EM-FRET hybrid method has mapped the Tfg2 C-terminus in the RNAPII-TFIIF and henceforth represents a novel and effective approach for elucidating the protein complex to nanometer precision.

Publication date: Available online 30 May 2013
Source:Journal of Structural Biology

Author(s): Benjamin T. Goult , Xiao-Ping Xu , Alexandre R. Gingras , Mark Swift , Bipin Patel , Neil Bate , Petra M. Kopp , Igor L. Barsukov , David R. Critchley , Niels Volkmann , Dorit Hanein

Talin is a large adaptor protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM (band 4.1, ezrin, radixin, moesin) domain (the head) linked to a flexible rod comprised of 13 amphipathic helical bundles (R1-R13) that terminate in a C-terminal helix (DD) that forms an anti-parallel dimer. We derived a three-dimensional structural model of full-length talin at a resolution of approximately 2.5 nm using EM reconstruction of full-length talin and the known shapes of the individual domains and inter-domain angles as derived from small angle X-ray scattering. Talin adopts a compact conformation consistent with a dimer in which the two talin rods forms a donut-shaped structure, with the two talin heads packed side by side occupying the hole at the center of this donut. In this configuration, the integrin binding site in the head domain and the actin-binding site at the carboxy-terminus of the rod are masked, implying that talin must unravel before it can support integrin activation and engage the actin cytoskeleton.

Publication date: Available online 29 May 2013
Source:Journal of Structural Biology

Author(s): Li Jiao , Jin-Sik Kim , Woo-Seok Song , Bo-Young Yoon , Kangseok Lee , Nam-Chul Ha

The disulfide-bond isomerase DsbC plays a crucial role in the folding of bacterial proteins in the periplasmic space. DsbC has a V-shaped dimeric structure with two domains, and Cys98 in the C-terminal domain attacks inappropriate disulfide bonds in substrate proteins due to its high nucleophilic activity. In this article, we present the crystal structure of DsbC from Salmonella enterica serovar Typhimurium. We evaluated the conserved residues Asp95 and Arg125, which are located close to Cys98. The mutation of Asp95 or Arg125 abolished the disulfide isomerase activity of DsbC in an in vitro assay using a protein substrate, and the R125A mutation significantly reduced the chaperone activity for the substrate RNase I in vivo. Furthermore, a comparative analysis suggested that the conformation of Arg125 varies depending on the packing or protein-protein interactions. Based on these findings, we suggest that Asp95 and Arg125 modulate the pKa of Cys98 during catalysis.

Publication date: Available online 25 May 2013
Source:Journal of Structural Biology

Author(s): Miloš Vulović , Raimond B.G. Ravelli , Lucas J. van Vliet , Abraham J. Koster , Ivan Lazić , Uwe Lücken , Hans Rullgård , Ozan Öktem , Bernd Rieger

Accurate modeling of image formation in cryo-electron microscopy is an important requirement for quantitative image interpretation and optimization of the data acquisition strategy. Here we present a forward model that accounts for the specimen’s scattering properties, microscope optics, and detector response. The specimen interaction potential is calculated with the isolated atom superposition approximation (IASA) and extended with the influences of solvent’s dielectric and ionic properties as well as the molecular electrostatic distribution. We account an effective charge redistribution via the Poisson-Boltzmann approach and find that the IASA-based potential forms the dominant part of the interaction potential, as the contribution of the redistribution is less than 10 %. The electron wave is propagated through the specimen by a multislice approach and the influence of the optics is included via the contrast transfer function. We incorporate the detective quantum efficiency of the camera due to the difference between signal and noise transfer characteristics, instead of using only the modulation transfer function. The full model was validated against experimental images of 20S proteasome, hemoglobin, and GroEL. The simulations adequately predict the effects of phase contrast, changes due to the integrated electron flux, thickness, inelastic scattering, detective quantum efficiency and acceleration voltage. We suggest that beam-induced specimen movements are relevant in the experiments whereas the influence of the solvent amorphousness can be neglected. All simulation parameters are based on physical principles and, when necessary, experimentally determined.

Publication date: Available online 24 May 2013
Source:Journal of Structural Biology

Author(s): Maggie Cusack , DuJiao Guo , Peter Chung , Nicholas A. Kamenos

The shell of the gastropod mollusc, abalone, is comprised of nacre with an outer prismatic layer that is composed of either calcite or aragonite or both, depending on the species. A striking characteristic of the abalone shell is the row of apertures along the dorsal margin. As the organism and shell grow, new apertures are formed and the preceding ones are filled in. Detailed investigations, using electron backscatter diffraction, of the infill in three species of abalone: Haliotis asinina, Haliotis gigantea and Haliotis rufescens reveals that, like the shell, the infill is composed mainly of nacre with an outer prismatic layer. The infill prismatic layer has identical mineralogy as the original shell prismatic layer. In H. asinina and H. gigantea, the prismatic layer of the shell and infill are made of aragonite while in H. rufescens both are composed of calcite. Abalone builds the infill material with the same high level of biological control, replicating the structure, mineralogy and crystallographic orientation as for the shell. The infill of abalone apertures presents us with insight into what is, effectively, shell repair.

Publication date: Available online 23 May 2013
Source:Journal of Structural Biology

Author(s): Ping-An Fang , Henry C. Margolis , James F. Conway , James P. Simmer , Elia Beniash

Amelogenin, the major extracellular enamel matrix protein, plays a critical role in regulating the growth and organization of enamel. Assembly and mineralization of full-length native (P173) and recombinant (rP172) porcine amelogenins were studied by cryogenic Transmission Electron Microscopy (cryoTEM). The cryoTEM revealed that both native and recombinant porcine amelogenins undergo step-wise self-assembly. Although the overall structural organization of P173 and rP172 oligomers was similar and resembled oligomers of murine recombinant amelogenin rM179, there were subtle differences suggesting that a single phosphorylated serine present in P173 might affect amelogenin self-assembly. Our mineralization studies demonstrated that both P173 and rP172 oligomers stabilize initial mineral clusters. Importantly, however, rP172 regulated the organization of initial mineral clusters into linear chains and guided the formation of parallel arrays of elongated mineral particles, which are the hallmark of enamel structural organization. These results are similar to those obtained previously using full-length recombinant murine amelogenin (Fang et al., 2011a). In contrast to that seen with rP172, phosphorylated P173 strongly inhibits mineralization for extended periods of time. We propose that these differences might be due to the differences in the structural organization and charge distribution between P173 and rP172. Overall our studies indicate that self-assembly of amelogenin and the mechanisms of its control over mineralization might be universal across different mammalian species. Our data also provide new insight into the effect of phosphorylation on amelogenin self-assembly and its regulation of mineralization.

Publication date: Available online 23 May 2013
Source:Journal of Structural Biology

Author(s): Benny Bar-On , H. Daniel Wagner

Recent progress made in the field of hierarchical biological materials is reviewed with an emphasis on the staggering characteristics at the smaller structural scale of a number of tissues. We show by means of selected examples that the small-scale architecture, and particularly the degree of staggering and overlap, plays a critical role in the macroscopic elastic behavior of those tissues.

Publication date: Available online 22 May 2013
Source:Journal of Structural Biology

Author(s): Hiroyuki Iwamoto

Insect flight muscle (IFM) can oscillate at frequencies up to 1000Hz, owing to its capability of stretch activation (SA). It is a highly specialized form of cross striated muscles, and its peculiar features include the IFM-specific isoform of troponin-I (troponin-H or TnH) with an unusually long Pro-Ala-rich extension at the C-terminus. Although we have shown that this extension does not directly take part in SA, questions remain as to what its real role is and why it is expressed only in IFM. Here we explored the structural role of the extension, be comparing X-ray diffraction patterns and electron micrographs of bumblebee IFM fibers before and after enzymatic removal of the extension. The removal had a dramatic effect on diffraction patterns: In IFMs in general, the equatorial 2,0 reflection is much stronger than the 1,1 reflection, but after removal, their intensities became almost equal (stronger 1,1 is a feature of vertebrate skeletal muscle). Electron micrographs revealed that a substantial fraction of the thin filaments showed a tendency to move towards the vertebrate position (the trigonal position between three thick filaments), while the rest of the thin filaments remained in their original insect position (midway between two neighboring thick filaments). Therefore, one of the roles of the extension is suggested to keep the filament lattice in the correct configuration for IFM. This insect-type lattice structure is preserved among IFMs from varied insect orders but not in body muscles, suggesting that the maintenance of this lattice structure is important for flight functions.

Publication date: Available online 21 May 2013
Source:Journal of Structural Biology

Author(s): Janne Ravantti , Dennis Bamford , David I. Stuart

The classification and alignment of multiple three-dimensional protein structures is a powerful way to detect similarities that cannot be discovered from the sequences alone and can help to infer phylogeny. However, the alignment process remains problematic for divergent structures. We have devised a fully automatic pipeline, HSF, drawing its inspiration from well-known structural alignment methods, which given a list of structures not only aligns all pairs but also classifies them fully. We demonstrate proof of principle for the new method by aligning the currently available set of highly diverged virus coat protein structures containing double β-barrels, as well as validating the method with established test sets for multiple structural alignments. The results for the virus proteins are inline with previous observations based on biochemical, genetic and structural studies but go further, since by providing coherent alignments between sets of molecules with marked structural distortion, they facilitate the marshaling of arguments for or against homology. The classification results can therefore be readily interpreted in terms of phylogeny.

Publication date: Available online 18 May 2013
Source:Journal of Structural Biology

Author(s): J. Bernard Heymann , Dennis C. Winkler , Yang-In Yim , Evan Eisenberg , Lois E. Greene , Alasdair C. Steven

Clathrin coats, which stabilize membrane curvature during endocytosis and vesicular trafficking, form highly polymorphic fullerene lattices. We used cryo-electron tomography to visualize coated particles in isolates from bovine brain. The particles range from ∼66 to ∼134nm in diameter, and only 20% of them (all ⩾80nm) contain vesicles. The remaining 80% are clathrin “baskets”, presumably artifactual assembly products. Polyhedral models were built for 54 distinct coat geometries. In true coated vesicles (CVs), most vesicles are offset to one side, leaving a crescent of interstitial space between the coat and the membrane for adaptor proteins and other components. The latter densities are fewer on the membrane-proximal side, which may represent the last part of the vesicle to bud off. A small number of densities – presumably cargo proteins – are associated with the interior surface of the vesicles. The clathrin coat, adaptor proteins, and vesicle membrane contribute almost all of the mass of a CV, with most cargoes accounting for only a few percent. The assembly of a CV therefore represents a massive biosynthetic effort to internalize a relatively diminutive payload. Such a high investment may be needed to overcome the resistance of membranes to high curvature.