nils-herrmann/clean_scirepeval huggingface dataset:
A method for 3D-reconstruction of a muscle thick filament using the tilt series images of a single filament electron tomogram.
Venue: Journal of structural biology

Myosin interacting-heads (MIH) motifs are visualized in 3D-reconstructions of thick filaments from striated muscle. These reconstructions are calculated by averaging methods using images from electron micrographs of grids prepared using numerous filament preparations. Here we propose an alternative method to calculate the 3D-reconstruction of a single thick filament using only a tilt series images recorded by electron tomography. Relaxed thick filaments, prepared from tarantula leg muscle homogenates, were negatively stained. Single-axis tilt series of single isolated thick filaments were obtained with the electron microscope at a low electron dose, and recorded on a CCD camera by electron tomography. An IHRSR 3D-recontruction was calculated from the tilt series images of a single thick filament. The reconstruction was enhanced by including in the search stage dual tilt image segments while only single tilt along the filament axis is usually used, as well as applying a band pass filter just before the back projection. The reconstruction from a single filament has a 40 Å resolution and clearly shows the presence of MIH motifs. In contrast, the electron tomogram 3D-reconstruction of the same thick filament - calculated without any image averaging and/or imposition of helical symmetry - only reveals MIH motifs infrequently. This is - to our knowledge - the first application of the IHRSR method to calculate a 3D reconstruction from tilt series images. This single filament IHRSR reconstruction method (SF-IHRSR) should provide a new tool to assess structural differences between well-ordered thick (or thin) filaments in a grid by recording separately their electron tomograms.
Identification of the chemical form of sulfur compounds in the Japanese pink coral (Corallium elatius) skeleton using μ-XRF/XAS speciation mapping.
Venue: Journal of structural biology

The distributions and chemical forms of sulfur compounds in the skeleton of Japanese pink coral (Corallium elatius) were investigated using X-ray spectroscopic techniques combined with micro-focused soft X-ray radiation. Microscopic X-ray fluorescence/soft X-ray photoabsorption (μ-XRF/XAS) speciation mapping clarified that sulfate is the primary species in the coral skeleton, with minor amounts of organic sulfur, whereas both sulfate and organic sulfur coexist in coenenchyme. Analysis of the post-edge region of the XAS spectra confirmed that sulfate ions in the coral skeleton are mainly in the form of gypsum-like inorganic sulfate substituting for the carbonate ions in the calcite skeleton. The sulfate concentration was negatively correlated with the magnesium concentration and positively correlated with that of phosphorus. Speciation mapping of sulfate in the coral skeleton showed clear fluctuations with sulfate concentrations being higher at dark bands, whereas the small amount of organic sulfur had unclear dark/bright bands. These results suggest that the little organic sulfur that is present is contained in the organic matter embedded in the biocrystal of coral skeleton.
A first census of collagen interruptions: collagen's own stutters and stammers.
Venue: Journal of structural biology

The repetitive Gly-X-Y sequence is the telltale sign of triple helical domains in collagens and collagen-like proteins. Most collagen sequences contain sporadic interruptions of this pattern, which may have functional roles in molecular flexibility, assembly or molecular recognition. However, the structural signatures of the different interruptions are not well defined. Here, a first comprehensive survey of collagen interruptions on collagen sequences from different taxonomic groups is presented. Amino acid preferences at the sites of interruption and the flanking triplets are analysed separately for metazoan and prokaryotic collagens and the concept of commensurateness between interruptions is introduced. Known structural information from model peptides is used to present a common framework for hydrogen bonding topology and variations in superhelical twist for the different types of interruptions. Several collagen interruptions are further classified here as stutters or stammers in analogy to the heptad breaks observed in alpha-helical coiled coils, and the structural consequences of commensurate interruptions in heterotrimeric collagens are briefly discussed. Data presented here will be useful for further investigation on the relation between structure and function of collagen interruptions.
Crystallinity and microchemistry of Nassarius reticulatus (Caenogastropoda) statoliths: towards their structure stability and homogeneity.
Venue: Journal of structural biology

Gastropod statoliths are spherical biocarbonates formed during their lifespan. The stability and homogeneity of these structures' mineral matrix was characterised along their radiuses, using Nassarius reticulatus as a model. Generally, they were proved to be bimineralic. Two of the three CaCO3 crystalline polymorphs occurring in biocarbonates - aragonite and calcite - coexist along statolith radiuses, aragonite being unequivocally the most abundant phase. The presence of a diffuse organic matrix was also perceived by the detection of a weak Raman band between 2800 and 3000 cm(-1) consistently observed along radiuses. Beyond the apparent stability and homogeneity, different crystalline orientations were disclosed by Raman spectroscopy. A change in the intensity pattern of the features related to the lattice and bending modes of aragonite between different radiuses give new insights for a possible spherulitic-like growth of these structures. As expected from the relative homogeneity of both mineral and organic signals, there was no pattern on the distribution of Ca, O, Na and S along radiuses. However, a higher concentration of Sr occurs in growth rings (known as winter tags), corroborating the already described negative correlation between the concentration of this element in statoliths and temperature. Despite the apparent stability and homogeneity of the matrix during its lifespan, the periodic distribution of Sr potentially influences a dissimilar incorporation of trace elements in increments and growth rings. Since gastropod statolith elemental fingerprinting was recently suggested as a new tool to monitor marine environmental changes, the pressing need for further studies on the incorporation of traces in these structures is highlighted.
The structure of AAVrh32.33, a novel gene delivery vector.
Venue: Journal of structural biology

The Adeno-associated viruses (AAVs) are being developed as gene delivery vectors for therapeutic clinical applications. However, the host antibody immune response directed against their capsid, prevalent in ∼40-70% of the general population, depending on serotype, negatively impacts efficacy. AAVrh32.33, a novel vector developed from rhesus macaques isolates, has significantly lower seroprevalence in human populations compared to AAV2 and AAV8, which are both in clinical use. To better understand the capsid determinants of this differential immune response to AAVrh32.33, its structure was determined by X-ray crystallography to 3.5 Å resolution. The capsid viral protein (VP) structure conserves the eight-stranded β-barrel core and αA helix reported for other parvoviruses and the distinct capsid surface topology of the AAVs: a depression at the icosahedral twofold axis, three protrusions surrounding the threefold axis, and a depression surround a cylindrical channel at the fivefold axis. A comparison to AAV2, AAV4, and AAV8, to which AAVrh32.33 shares ∼61%, ∼81%, and ∼63% identity, respectively, identified differences in previously defined AAV VP structurally variable regions (VR-1 to VR-IX) which function as receptor attachment, transduction efficiency, and/or antigenic determinants. This structure thus provides a 3D platform for capsid engineering in ongoing efforts to develop AAVrh32.33, as well as other AAV serotypes, for tissue targeted gene-therapy applications with vectors that can evade pre-existing antibody responses against the capsid. These features are required for full clinical realization of the promising AAV gene delivery system.
Vitrification of Tokuyasu-style immuno-labelled sections for correlative cryo light microscopy and cryo electron tomography.
Venue: Journal of structural biology

We present an approach for the preparation of immuno-labelled ultrathin sections from cells or tissue that are compatible with both fluorescence and transmission electron microscopy. Our approach is inspired by a method of Sabanay et al. (1991) that is based on the Tokuyasu technique for immunogold labelling of sections from aldehyde-fixed samples. The difference of this method with the original Tokuyasu technique is that the immuno-labelled sections are stabilized in a thin layer of vitreous water by plunge-freezing prior to electron microscopical observation. The vitrification step allows for phase contrast-based imaging at cryogenic conditions. We show that this immuno-labelling method is well-suited for imaging cellular ultrastructure in three dimensions (tomography) at cryogenic conditions, and that fluorescence associated with the sections is retained. This method is a valuable tool for Correlative Light and Electron Microscopy (CLEM), and we refer to this method in combination with CLEM as VOS (vitrification of sections). We provide examples for the application of VOS using dendritic cells and neurons, and show specifically that this method enables the researcher to navigate to lysosomes and synapses.
Correlative light- and electron microscopy with chemical tags.
Venue: Journal of structural biology

Correlative microscopy incorporates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Several approaches exist for correlative microscopy, most of which have used the green fluorescent protein (GFP) as the label for light microscopy. Here we use chemical tagging and synthetic fluorophores instead, in order to achieve protein-specific labeling, and to perform multicolor imaging. We show that synthetic fluorophores preserve their post-embedding fluorescence in the presence of uranyl acetate. Post-embedding fluorescence is of such quality that the specimen can be prepared with identical protocols for scanning electron microscopy (SEM) and transmission electron microscopy (TEM); this is particularly valuable when singular or otherwise difficult samples are examined. We show that synthetic fluorophores give bright, well-resolved signals in super-resolution light microscopy, enabling us to superimpose light microscopic images with a precision of up to 25 nm in the x-y plane on electron micrographs. To exemplify the preservation quality of our new method we visualize the molecular arrangement of cadherins in adherens junctions of mouse epithelial cells.
Automated particle correspondence and accurate tilt-axis detection in tilted-image pairs.
Venue: Journal of structural biology

Tilted electron microscope images are routinely collected for an ab initio structure reconstruction as a part of the Random Conical Tilt (RCT) or Orthogonal Tilt Reconstruction (OTR) methods, as well as for various applications using the "free-hand" procedure. These procedures all require identification of particle pairs in two corresponding images as well as accurate estimation of the tilt-axis used to rotate the electron microscope (EM) grid. Here we present a computational approach, PCT (particle correspondence from tilted pairs), based on tilt-invariant context and projection matching that addresses both problems. The method benefits from treating the two problems as a single optimization task. It automatically finds corresponding particle pairs and accurately computes tilt-axis direction even in the cases when EM grid is not perfectly planar.
Crystallographic snapshot of the Escherichia coli EnvZ histidine kinase in an active conformation.
Venue: Journal of structural biology

Sensor histidine kinases are important sensors of the extracellular environment and relay signals via conformational changes that trigger autophosphorylation of the kinase and subsequent phosphorylation of a response regulator. The exact mechanism and the regulation of this protein family are a matter of ongoing investigation. Here we present a crystal structure of a functional chimeric protein encompassing the entire catalytic part of the Escherichia coli EnvZ histidine kinase, fused to the HAMP domain of the Archaeoglobus fulgidus Af1503 receptor. The construct is thus equivalent to the full cytosolic part of EnvZ. The structure shows a putatively active conformation of the catalytic domain and gives insight into how this conformation could be brought about in response to sensory input. Our analysis suggests a sequential flip-flop autokinase mechanism.
TRDistiller: a rapid filter for enrichment of sequence datasets with proteins containing tandem repeats.
Venue: Journal of structural biology

The dramatic growth of sequencing data evokes an urgent need to improve bioinformatics tools for large-scale proteome analysis. Over the last two decades, the foremost efforts of computer scientists were devoted to proteins with aperiodic sequences having globular 3D structures. However, a large portion of proteins contain periodic sequences representing arrays of repeats that are directly adjacent to each other (so called tandem repeats or TRs). These proteins frequently fold into elongated fibrous structures carrying different fundamental functions. Algorithms specific to the analysis of these regions are urgently required since the conventional approaches developed for globular domains have had limited success when applied to the TR regions. The protein TRs are frequently not perfect, containing a number of mutations, and some of them cannot be easily identified. To detect such "hidden" repeats several algorithms have been developed. However, the most sensitive among them are time-consuming and, therefore, inappropriate for large scale proteome analysis. To speed up the TR detection we developed a rapid filter that is based on the comparison of composition and order of short strings in the adjacent sequence motifs. Tests show that our filter discards up to 22.5% of proteins which are known to be without TRs while keeping almost all (99.2%) TR-containing sequences. Thus, we are able to decrease the size of the initial sequence dataset enriching it with TR-containing proteins which allows a faster subsequent TR detection by other methods. The program is available upon request.
Axial helix rotation as a mechanism for signal regulation inferred from the crystallographic analysis of the E. coli serine chemoreceptor.
Venue: Journal of structural biology

Bacterial chemotaxis receptors are elongated homodimeric coiled-coil bundles, which transduce signals generated in an N-terminal sensor domain across 15-20nm to a conserved C-terminal signaling subdomain. This signal transduction regulates the activity of associated kinases, altering the behavior of the flagellar motor and hence cell motility. Signaling is in turn modulated by selective methylation and demethylation of specific glutamate and glutamine residues in an adaptation subdomain. We have determined the structure of a chimeric protein, consisting of the HAMP domain from Archaeoglobus fulgidus Af1503 and the methyl-accepting domain of Escherichia coli Tsr. It shows a 21nm coiled coil that alternates between two coiled-coil packing modes: canonical knobs-into-holes and complementary x-da, a variant form related to the canonical one by axial rotation of the helices. Comparison of the obtained structure to the Thermotoga maritima chemoreceptor TM1143 reveals that they adopt different axial rotation states in their adaptation subdomains. This conformational change is presumably induced by the upstream HAMP domain and may modulate the affinity of the chemoreceptor to the methylation-demethylation system. The presented findings extend the cogwheel model for signal transmission to chemoreceptors.
A structural analysis of the AAA+ domains in Saccharomyces cerevisiae cytoplasmic dynein
Venue: Journal of structural biology

Dyneins are large protein complexes that act as microtubule based molecular motors. The dynein heavy chain contains a motor domain which is a member of the AAA+ protein family (ATPases Associated with diverse cellular Activities). Proteins of the AAA+ family show a diverse range of functionalities, but share a related core AAA+ domain, which often assembles into hexameric rings. Dynein is unusual because it has all six AAA+ domains linked together, in one long polypeptide. The dynein motor domain generates movement by coupling ATP driven conformational changes in the AAA+ ring to the swing of a motile element called the linker. Dynein binds to its microtubule track via a long antiparallel coiled-coil stalk that emanates from the AAA+ ring. Recently the first high resolution structures of the dynein motor domain were published. Here we provide a detailed structural analysis of the six AAA+ domains using our Saccharomycescerevisiae crystal structure. We describe how structural similarities in the dynein AAA+ domains suggest they share a common evolutionary origin. We analyse how the different AAA+ domains have diverged from each other. We discuss how this is related to the function of dynein as a motor protein and how the AAA+ domains of dynein compare to those of other AAA+ proteins.
2dx_automator: implementation of a semiautomatic high-throughput high-resolution cryo-electron crystallography pipeline.
Venue: Journal of structural biology

The introduction of direct electron detectors (DED) to cryo-electron microscopy has tremendously increased the signal-to-noise ratio (SNR) and quality of the recorded images. We discuss the optimal use of DEDs for cryo-electron crystallography, introduce a new automatic image processing pipeline, and demonstrate the vast improvement in the resolution achieved by the use of both together, especially for highly tilted samples. The new processing pipeline (now included in the software package 2dx) exploits the high SNR and frame readout frequency of DEDs to automatically correct for beam-induced sample movement, and reliably processes individual crystal images without human interaction as data are being acquired. A new graphical user interface (GUI) condenses all information required for quality assessment in one window, allowing the imaging conditions to be verified and adjusted during the data collection session. With this new pipeline an automatically generated unit cell projection map of each recorded 2D crystal is available less than 5 min after the image was recorded. The entire processing procedure yielded a three-dimensional reconstruction of the 2D-crystallized ion-channel membrane protein MloK1 with a much-improved resolution of 5Å in-plane and 7Å in the z-direction, within 2 days of data acquisition and simultaneous processing. The results obtained are superior to those delivered by conventional photographic film-based methodology of the same sample, and demonstrate the importance of drift-correction.
Frealix: model-based refinement of helical filament structures from electron micrographs.
Venue: Journal of structural biology

The structures of many helical protein filaments can be derived from electron micrographs of their suspensions in thin films of vitrified aqueous solutions. The most successful and generally-applicable approach treats short segments of these filaments as independent "single particles", yielding near-atomic resolution for rigid and well-ordered filaments. The single-particle approach can also accommodate filament deformations, yielding sub-nanometer resolution for more flexible filaments. However, in the case of thin and flexible filaments, such as some amyloid-β (Aβ) fibrils, the single-particle approach may fail because helical segments can be curved or otherwise distorted and their alignment can be inaccurate due to low contrast in the micrographs. We developed new software called Frealix that allows the use of arbitrarily short filament segments during alignment to approximate even high curvatures. All segments in a filament are aligned simultaneously with constraints that ensure that they connect to each other in space to form a continuous helical structure. In this paper, we describe the algorithm and benchmark it against datasets of Aβ(1-40) fibrils and tobacco mosaic virus (TMV), both analyzed in earlier work. In the case of TMV, our algorithm achieves similar results to single-particle analysis. In the case of Aβ(1-40) fibrils, we match the previously-obtained resolution but we are also able to obtain reliable alignments and ∼8-Å reconstructions from curved filaments. Our algorithm also offers a detailed characterization of filament deformations in three dimensions and enables a critical evaluation of the worm-like chain model for biological filaments.
Influence of repeat numbers on self-assembly rates of repetitive recombinant spider silk proteins.
Venue: Journal of structural biology

Assembly of recombinant spider silk variants eADF4(Cn) comprising different numbers (n) of the consensus sequence motif C, derived from the natural Araneus diadematus dragline silk ADF4, yielded indistinguishable nanofibrils in cases of n⩾2. The C-module comprises 35 amino acids rich in glycine and proline residues (in GPGXY repeats) and one polyalanine stretch (Ala)8. All variants were found to be intrinsically disordered in solution, and upon fibril formation they converted into a cross-β structure. Heterologous seeding indicated high structural compatibility between the different eADF4(Cn) variants, however, their assembly kinetics differed in dependence of the number of repeats. Kinetic analysis revealed a nucleation-growth mechanism typical for the formation of cross-β-fibrils, with nucleation rates as well as growth rates increasing with increasing numbers of repeats. Strikingly, the single C-module did not self-assemble into fibrils, but upon addition of heterologous seeds fibril growth could be observed. Apparently, interconnecting of at least two C-modules significantly facilitates the structural transformation from a disordered state into β-sheet structures, which is necessary for nucleation and beneficial for fibril growth.
The spectrin family of proteins: a unique coiled-coil fold for various molecular surface properties.
Venue: Journal of structural biology

The spectrin superfamily is composed of proteins involved in cytolinker functions. Their main structural feature is a large central subdomain with numerous repeats folded in triple helical coiled-coils. Their similarity of sequence was considered to be low without detailed quantification of the intra- and intermolecular levels. Among the superfamily, we considered as essential to propose an overview of the surface properties of all the repeats of the five proteins of the spectrin family, namely α- and β-spectrins, α-actinin, dystrophin and utrophin. Therefore, the aim of this work was to obtain a quantitative comparison of all the repeats at both the primary sequence and the three-dimensional levels. For that purpose, we applied homology modelling methods to obtain structural models for successive and overlapping tandem repeats of the human erythrocyte α- and β-spectrins and utrophin, as previously undertaken for dystrophin, and we used the known structure of α-actinin. The matrix calculation of the pairwise similarities of all the repeat sequences and the electrostatic and hydrophobic surface properties throughout the protein family support the view that spectrins and α-actinin on one hand and utrophin and dystrophin on the other hand share some structural similarities, but a detailed molecular characterisation highlights substantial differences. The repeats within the family are far from identical, which is consistent with their multiple interactions with different cellular partners, including proteins and membrane lipids.