nils-herrmann/clean_scirepeval huggingface dataset:
Deducing the symmetry of helical assemblies: Applications to membrane proteins.
Venue: Journal of structural biology

Helical reconstruction represents a convenient and powerful approach for structure determination of macromolecules that assemble into helical arrays. In the case of membrane proteins, formation of tubular crystals with helical symmetry represents an attractive alternative, especially when their small size precludes the use of single-particle analysis. An essential first step for helical reconstruction is to characterize the helical symmetry. This process is often daunting, due to the complexity of helical diffraction and to the low signal-to-noise ratio in images of individual assemblies. Furthermore, the large diameters of the tubular crystals produced by membrane proteins exacerbates the innate ambiguities that, if not resolved, will produce incorrect structures. In this report, we describe a set of tools that can be used to eliminate ambiguities and to validate the choice of symmetry. The first approach increases the signal-to-noise ratio along layer lines by incoherently summing data from multiple helical assemblies, thus producing several candidate indexing schemes. The second approach compares the layer lines from images with those from synthetic models built with the various candidate schemes. The third approach uses unit cell dimensions measured from collapsed tubes to distinguish between these candidate schemes. These approaches are illustrated with tubular crystals from a boron transporter from yeast, Bor1p, and a β-barrel channel from the outer membrane of E. coli, OmpF.
Insights into autophagosome biogenesis from in vitro reconstitutions
Venue: Journal of structural biology

Macro-autophagy (autophagy) is a conserved catabolic pathway for the degradation of cytoplasmic material in the lysosomal system. This is achieved by the sequestration of the cytoplasmic cargo material within double membrane-bound vesicles that fuse with lysosomes, wherein the vesicle’s inner membrane and the cargo are degraded. Autophagosomes form in a de novo manner and their precursors are initially detected as small membrane structures that are referred to as isolation membranes. The isolation membranes gradually expand and subsequently close to give rise to autophagosomes. Many proteins required to form autophagosomes have been identified but how they act mechanistically is still enigmatic. Here we critically review reconstitution approaches employed to decipher the inner working of the fascinating autophagy machinery.
Intrinsic aggregation propensity of the CsgB nucleator protein is crucial for curli fiber formation.
Venue: Journal of structural biology

Several organisms exploit the extraordinary physical properties of amyloid fibrils forming natural protective amyloids, in an effort to support complex biological functions. Curli amyloid fibers are a major component of mature biofilms, which are produced by many Enterobacteriaceae species and are responsible, among other functions, for the initial adhesion of bacteria to surfaces or cells. The main axis of curli fibers is formed by a major structural subunit, known as CsgA. CsgA self-assembly is promoted by oligomeric nuclei formed by a minor curli subunit, known as the CsgB nucleator protein. Here, by implementing AMYLPRED2, a consensus prediction method for the identification of 'aggregation-prone' regions in protein sequences, developed in our laboratory, we have successfully identified potent amyloidogenic regions of the CsgB subunit. Peptide-analogues corresponding to the predicted 'aggregation-prone' segments of CsgB were chemically synthesized and studied, utilizing several biophysical techniques. Our experimental data indicate that these peptides self-assemble in solution, forming fibrils with characteristic amyloidogenic properties. Using comparative modeling techniques, we have developed three-dimensional models of both CsgA and CsgB subunits. Structural analysis revealed that the identified 'aggregation-prone' segments may promote gradual polymerization of CsgB. Briefly, our results indicate that the intrinsic self-aggregation propensity of the CsgB subunit, most probably has a pivotal role in initiating the formation of curli amyloid fibers by promoting the self-assembly process of the CsgB nucleator protein.
Subtomogram analysis using the Volta phase plate.
Venue: Journal of structural biology

Cryo-electron tomography (CET) and subtomogram analysis allow studying the structures of macromolecular complexes in their natural context. The radiation sensitivity of vitrified biological specimens and the resulting low signal-to-noise ratio (SNR) in CET limit the amount of structural information that can be mined from tomographic data. The Volta phase plate (VPP) has emerged as an effective means to increase the SNR and hence contrast compared to 'conventional' defocus-based phase contrast transmission electron microscopy (CTEM). Here, we assess the performance of the VPP compared to CTEM in subtomogram analysis, using the mammalian 80S ribosome as a test case. Accurate focusing is the major factor for achieving high resolution with the VPP, as highlighted by a comparison of slightly different focusing strategies. From only 1400 subtomograms, the VPP yields a subtomogram average of the mammalian 80S ribosome at 9.6Å resolution without laborious contrast transfer function (CTF) correction. The subtomogram averages obtained using CTEM approaches are comparable, but suffer from lower signal transfer in certain frequency bands due to the oscillations of the CTF. Our study demonstrates that the VPP is a valuable tool for subtomogram analysis, because it enables improved performance and efficiency in terms of structure localization and number of subtomograms required for a given resolution.
A multilayer micromechanical model of the cuticle of Curculio longinasus Chittenden, 1927 (Coleoptera: Curculionidae).
Venue: Journal of structural biology

Curculio longinasus Chittenden, 1927 (Coleoptera: Curculionidae), is a weevil species common throughout the southwestern United States that uses its rostrum - a very slender, curved, beak-like projection of the head - to excavate tunnels in plant organs (such as acorns) for egg laying (oviposition). Once the apical portion of the rostrum has been inserted into the preferred substrate for oviposition, the female begins rotating around the perimeter of the hole, elevating her head by extending the fore-legs, and rotating the head in place in a drilling motion. This action causes significant elastic deformation of the rostrum, which will bend until it becomes completely straight. To better understand the mechanical behavior of the cuticle as it undergoes deformation during the preparation of oviposition sites, we develop a comprehensive micro/macro model of the micromechanical structure and properties of the cuticle, spanning across all cuticular regions, and reliably mirroring the resultant macroscale properties of the cuticle. Our modeling approach relies on the use of multi-scale, hierarchical biomaterial representation, and employs various micromechanical schemata - e.g., Mori-Tanaka, effective field, and Maxwell - to calculate the homogenized properties of representative volume elements at each level in the hierarchy. We describe the configuration and behavior of this model in detail, and discuss the theoretical implications and limitations of this approach with emphasis on future biomechanical and comparative evolutionary research. Our detailed account of this approach can thereby serve as a methodological template for exploring the biomechanical behavior of new insect structures.
Contributions of the lower dimer to supramolecular actin patterning revealed by TIRF microscopy.
Venue: Journal of structural biology

Two distinct dimers are formed during the initial steps of actin polymerization. The first one, referred to as the 'lower dimer' (LD) was discovered many years ago by means of chemical crosslinking. Owing to its transient nature, a biological relevance had long been precluded when, using LD-specific antibodies, we detected LD-like contacts in actin assemblies that are associated with the endolysosomal compartment in a number of different cell lines. Moreover, immunofluorescence showed the presence of LD-related structures at the cell periphery of migrating fibroblasts, in the nucleus, and in association with the centrosome of interphase cells. Here, we explore contributions of the LD to the assembly of supramolecular actin structures in real time by total internal reflection fluorescence (TIRF) microscopy. Our data shows that while LD on its own cannot polymerize under filament forming conditions, it is able to incorporate into growing F-actin filaments. This incorporation of LD triggers the formation of X-shaped filament assemblies with barbed ends that are pointing in the same direction in the majority of cases. Similarly, an increased frequency of junction sites was observed when filaments were assembled in the presence of oxidized actin. This data suggests that a disulfide bridge between Cys374 residues might stabilize LD-contacts. Based on our findings, we propose two possible models for the molecular mechanism underlying the supramolecular actin patterning in LD-related structures.
Detailed spectroscopic study of the role of Br and Sr in coloured parts of the Callinectes sapidus crab claw.
Venue: Journal of structural biology

The exoskeleton of crustaceans consists mainly of calcium carbonate (CaCO3) minerals and in many cases exhibits vivid colouration due to the presence of proteins rich in carotenoid chromophores. The exposure of aquatic animals in sea water results often in the incorporation of trace elements in their exoskeleton. The bonding configuration of Br and Sr trace elements in regions with different staining (white, orange and blue) of the exoskeleton of the Callinectes sapidus in crab claw are systematically investigated by a number of complementary spectroscopic techniques, including X-ray absorption fine structure spectroscopy (EXAFS), X-ray fluorescence, Raman and visible light reflectivity spectroscopies. It is found that Sr substitutes for Ca and the Sr/Ca ratio is constant along the claw. In the orange region that includes the claw fingers, CaCO3 adopts a calcite-like structure, whereas in the blue and white regions, located in the palm of the claw, an aragonite-like structure dominates. On the other hand, Br, present only in the blue and orange stained parts of the claw, is bound to phenyl and/or phenol rings of amino acid residues, most probably to phenylalanine and/or tyrosine, of the chromophore protein.
Structure of USP7 catalytic domain and three Ubl-domains reveals a connector α-helix with regulatory role.
Venue: Journal of structural biology

Ubiquitin conjugation is an important signal in cellular pathways, changing the fate of a target protein, by degradation, relocalisation or complex formation. These signals are balanced by deubiquitinating enzymes (DUBs), which antagonize ubiquitination of specific protein substrates. Because ubiquitination pathways are critically important, DUB activity is often carefully controlled. USP7 is a highly abundant DUB with numerous targets that plays complex roles in diverse pathways, including DNA regulation, p53 stress response and endosomal protein recycling. Full-length USP7 switches between an inactive and an active state, tuned by the positioning of 5 Ubl folds in the C-terminal HUBL domain. The active state requires interaction between the last two Ubls (USP7(45)) and the catalytic domain (USP7(CD)), and this can be promoted by allosteric interaction from the first 3 Ubl domains of USP7 (USP7(123)) interacting with GMPS. Here we study the transition between USP7 states. We provide a crystal structure of USP7(CD123) and show that CD and Ubl123 are connected via an extended charged alpha helix. Mutational analysis is used to determine whether the charge and rigidity of this 'connector helix' are important for full USP7 activity.
The unexpected structure of the designed protein Octarellin V.1 forms a challenge for protein structure prediction tools.
Venue: Journal of structural biology

Despite impressive successes in protein design, designing a well-folded protein of more 100 amino acids de novo remains a formidable challenge. Exploiting the promising biophysical features of the artificial protein Octarellin V, we improved this protein by directed evolution, thus creating a more stable and soluble protein: Octarellin V.1. Next, we obtained crystals of Octarellin V.1 in complex with crystallization chaperons and determined the tertiary structure. The experimental structure of Octarellin V.1 differs from its in silico design: the (αβα) sandwich architecture bears some resemblance to a Rossman-like fold instead of the intended TIM-barrel fold. This surprising result gave us a unique and attractive opportunity to test the state of the art in protein structure prediction, using this artificial protein free of any natural selection. We tested 13 automated webservers for protein structure prediction and found none of them to predict the actual structure. More than 50% of them predicted a TIM-barrel fold, i.e. the structure we set out to design more than 10years ago. In addition, local software runs that are human operated can sample a structure similar to the experimental one but fail in selecting it, suggesting that the scoring and ranking functions should be improved. We propose that artificial proteins could be used as tools to test the accuracy of protein structure prediction algorithms, because their lack of evolutionary pressure and unique sequences features.
Organelle remodeling at membrane contact sites.
Venue: Journal of structural biology

Cellular organelles must execute sophisticated biological processes to persist, and often communicate with one another to exchange metabolites and information. Recent studies suggest inter-organelle membrane contact sites (MCSs) are hubs for this cellular cross-talk. MCSs also govern membrane remodeling, thus controlling aspects of organelle shape, identity, and function. Here, we summarize three emerging phenomena that MCSs appear to govern: 1) organelle identity via the non-vesicular exchange of lipids, 2) mitochondrial shape and division, and 3) endosomal migration in response to sterol trafficking. We also discuss the role for ER-endolysosomal contact sites in cholesterol metabolism, and the potential biomedical importance this holds. Indeed, the emerging field inter-organellar cross-talk promises substantial advances in the fields of lipid metabolism and cell signaling.
Pf-Sp8/9, a novel member of the specificity protein family in Pinctada fucata, potentially participates in biomineralization.
Venue: Journal of structural biology

Specificity protein (Sp) belong to a transcription factor family that contains nine subgroups with essential functions in development, including skeletogenesis, tooth development, neural tube closure, and limb formation. In molluscs, functions of the Sp protein family members have not been reported in detail. In this study, we report the first Sp protein-encoding gene in Pinctada fucata. We named the translated protein Pf-Sp8/9, based on the phylogenetic development tree constructed using Sp protein sequences from six model organisms, which showed that it was a Sp8/9 homolog. Alignment of the Pf-Sp8/9 sequence with the amino acid sequences of related proteins showed that Pf-Sp8/9 had conserved domains, including three DNA-binding motifs. The tissue distribution showed that while Pf-Sp8/9 mRNA expression was detected in all tested tissues, it was particularly high in the mantle. The luciferase reporter assay results showed that Pf-Sp8/9 had the ability to activate the transcription of a number of matrix proteins. The expression pattern of Pf-Sp8/9 during P. fucata pearl sac development was similar to that of some genes that encode matrix proteins, suggesting Pf-Sp8/9 may be involved in mantle-related physiological activities and biomineralization.
An improved high pressure freezing and freeze substitution method to preserve the labile vaccinia virus nucleocapsid.
Venue: Journal of structural biology

In recent years, high pressure freezing and freeze substitution have been widely used for electron microscopy to reveal viral and cellular structures that are difficult to preserve. Vaccinia virus, a member of the Poxviridae family, presents one of the most complex viral structures. The classical view of vaccinia virus structure consists of an envelope surrounding a biconcave core, with a lateral body in each concavity of the core. This classical view was challenged by Peters and Muller (1963), who demonstrated the presence of a folded tubular structure inside the virus core and stated the difficulty in visualizing this structure, possibly because it is labile and cannot be preserved by conventional sample preparation. Therefore, this tubular structure, now called the nucleocapsid, has been mostly neglected over the years. Earlier studies were able to preserve the nucleocapsid, but with low efficiency. In this study, we report the protocol (and troubleshooting) that resulted in preservation of the highest numbers of nucleocapsids in several independent preparations. Using this protocol, we were able to demonstrate an interdependence between the formation of the virus core wall and the nucleocapsid, leading to the hypothesis that an interaction exists between the major protein constituents of these compartments, A3 (core wall) and L4 (nucleocapsid). Our results show that high pressure freezing and freeze substitution can be used in more in-depth studies concerning the nucleocapsid structure and function.
Ultrastructural aspects of foreign body giant cells generated on different substrates.
Venue: Journal of structural biology

Implantation of biomaterials into the body, e.g. for tissue engineering purposes, induces a material-dependent inflammatory response called the foreign body reaction (FBR). A hallmark feature of this response is the formation of large multinucleated cells: foreign body giant cells (FBGCs). Biomaterials like cross-linked and non-cross-linked collagen often induce the formation of FBGCs. It is unknown whether different biomaterials result in the formation of different FBGCs. To investigate this, we implanted cross-linked and non-cross-linked dermal sheep collagen subcutaneously in mice. After 21 days the implanted material was collected and prepared for ultrastructural analysis. More FBGCs formed on and between implants of cross-linked collagen compared to non-cross-linked material. The ultrastructural aspects of the FBGCs present on the two types of implants proved to be similar. On both materials, they formed long slender protrusions on the basolateral membrane, they were very rich in mitochondria, contained numerous nuclei, and showed signs of the presence of a clear zone facing the implanted material. Similar clear zones, that resemble osteoclastic features, were also seen in FBGCs generated in vitro on bone slices, but these cells did not form a ruffled border. However, similarities in ultrastructure such as the occurrence of slender protrusions and high mitochondrion content were also found in the FBGCs generated in vitro. These data indicate that FBGCs formed on different substrates share many morphological characteristics. The formation of long finger-like protrusions seemed typical for the FBGCs, in vivo as well as in vitro, however the function of these structures needs further analysis.
FIRT: Filtered iterative reconstruction technique with information restoration.
Venue: Journal of structural biology

Electron tomography (ET) combining subsequent sub-volume averaging has been becoming a unique way to study the in situ 3D structures of macromolecular complexes. However, information missing in electron tomography due to limited angular sampling is still the bottleneck in high-resolution electron tomography application. Here, based on the understanding of smooth nature of biological specimen, we present a new iterative image reconstruction algorithm, FIRT (filtered iterative reconstruction technique) for electron tomography by combining the algebra reconstruction technique (ART) and the nonlinear diffusion (ND) filter technique. Using both simulated and experimental data, in comparison to ART and weight back projection method, we proved that FIRT could generate a better reconstruction with reduced ray artifacts and significant improved correlation with the ground truth and partially restore the information at the non-sampled angular region, which was proved by investigating the 90° re-projection and by the cross-validation method. This new algorithm will be subsequently useful in the future for both cellular and molecular ET with better quality and improved structural details.
Denoising and Covariance Estimation of Single Particle Cryo-EM Images
Venue: Journal of structural biology

The problem of image restoration in cryo-EM entails correcting for the effects of the Contrast Transfer Function (CTF) and noise. Popular methods for image restoration include 'phase flipping', which corrects only for the Fourier phases but not amplitudes, and Wiener filtering, which requires the spectral signal to noise ratio. We propose a new image restoration method which we call 'Covariance Wiener Filtering' (CWF). In CWF, the covariance matrix of the projection images is used within the classical Wiener filtering framework for solving the image restoration deconvolution problem. Our estimation procedure for the covariance matrix is new and successfully corrects for the CTF. We demonstrate the efficacy of CWF by applying it to restore both simulated and experimental cryo-EM images. Results with experimental datasets demonstrate that CWF provides a good way to evaluate the particle images and to see what the dataset contains even without 2D classification and averaging.
Pushing the size limit of de novo structure ensemble prediction guided by sparse SDSL-EPR restraints to 200 residues: The monomeric and homodimeric forms of BAX.
Venue: Journal of structural biology

Structure determination remains a challenge for many biologically important proteins. In particular, proteins that adopt multiple conformations often evade crystallization in all biologically relevant states. Although computational de novo protein folding approaches often sample biologically relevant conformations, the selection of the most accurate model for different functional states remains a formidable challenge, in particular, for proteins with more than about 150 residues. Electron paramagnetic resonance (EPR) spectroscopy can obtain limited structural information for proteins in well-defined biological states and thereby assist in selecting biologically relevant conformations. The present study demonstrates that de novo folding methods are able to accurately sample the folds of 192-residue long soluble monomeric Bcl-2-associated X protein (BAX). The tertiary structures of the monomeric and homodimeric forms of BAX were predicted using the primary structure as well as 25 and 11 EPR distance restraints, respectively. The predicted models were subsequently compared to respective NMR/X-ray structures of BAX. EPR restraints improve the protein-size normalized root-mean-square-deviation (RMSD100) of the most accurate models with respect to the NMR/crystal structure from 5.9Å to 3.9Å and from 5.7Å to 3.3Å, respectively. Additionally, the model discrimination is improved, which is demonstrated by an improvement of the enrichment from 5% to 15% and from 13% to 21%, respectively.