nils-herrmann/clean_scirepeval huggingface dataset:
Solution structure of the cyclic-nucleotide binding homology domain of a KCNH channel.
Venue: Journal of structural biology

The carboxy-terminal region of the KCNH family of potassium channels contains a cyclic-nucleotide binding homology domain (CNBHD) that is important for channel gating and trafficking. The solution structure of the CNBHD of the KCNH potassium of zebrafish was determined using solution NMR spectroscopy. This domain exists as a monomer under solution conditions and adopts a similar fold to that determined by X-ray crystallography. The CNBHD does not bind cAMP because residue Y740 blocks the entry of cyclic-nucleotide to the binding pocket. Relaxation results show that the CNBHD is rigid except that some residues in the loop between β6 and β7 are flexible. Our results will be useful to understand the gating mechanism of KCNH family members through the CNBHD.
Three-dimensional structure of minipig fibrolamellar bone: adaptation to axial loading.
Venue: Journal of structural biology

Fibrolamellar bone is transiently produced by large, fast growing mammals. The fibrolamellar bone unit is initially formed by elaboration of a network of blood vessels. This is followed by the deposition of a thin, porous and hypercalcified layer, then by the infilling of the vascular cavities by the sequential deposition of a relatively thick rapidly forming bone on both sides of the hypercalcified layer, and finally by lamellar bone. We investigated the 3D structure of the collagenous network of fibrolamellar bone from the femora of a young minipig using mainly the FIB-SEM dual beam microscope and the Serial Surface View method. This enabled us to identify the fibril orientation, the canalicular network organization and other structural motifs within each element of the fibrolamellar unit. The first formed primary hypercalcified layer (PHL) is composed of fibril arrays and multiple small pores, and appears to have an isotropic structure. The major bone component is deposited on both sides of the PHL, and is composed of collagen fibrils with a preferred orientation, mainly aligned parallel to the bone long axis. This bone component is therefore parallel-fibered bone and not woven bone. We also observed that the collagen fibers are organized into bundles. The lamellar bone has most of its collagen fibrils aligned with the bone long axis. This study therefore shows that the large majority of collagen fibrils in fibrolamellar bone are aligned with the bone long axis. This anisotropic structure therefore appears to be adapted to loading along the bone long axis.
Local regularization of tilt projections reduces artifacts in electron tomography.
Venue: Journal of structural biology

Electron tomography produces very high resolution 3D image volumes useful for investigating the structure and function of cellular components. Unfortunately, unavoidable discontinuities and physical constraints in the acquisition geometry lead to a range of artifacts that can affect the reconstructed image. In particular, highly electron dense regions, such as gold nanoparticles, can hide proximal biological structures and degrade the overall quality of the reconstructed tomograms. In this work we introduce a pre-reconstruction non-conservative non-linear isotropic diffusion (NID) filter that automatically identifies and reduces local irregularities in the tilt projections. We illustrate the improvement in quality obtained using this approach for reconstructed tomograms generated from samples of malaria parasite-infected red blood cells. A quantitative and qualitative evaluation for our approach on both simulated and real data is provided.
Improving coiled coil stability while maintaining specificity by a bacterial hitchhiker selection system.
Venue: Journal of structural biology

The design and selection of peptides targeting cellular proteins is challenging and often yields candidates with undesired properties. Therefore we deployed a new selection system based on the twin-arginine translocase (TAT) pathway of Escherichia coli, named hitchhiker translocation (HiT) selection. A pool of α-helix encoding sequences was designed and selected for interference with the coiled coil domain (CC) of a melanoma-associated basic-helix-loop-helix-leucine-zipper (bHLHLZ) protein, the microphthalmia associated transcription factor (MITF). One predominant sequence (iM10) was enriched during selection and showed remarkable protease resistance, high solubility and thermal stability while maintaining its specificity. Furthermore, it exhibited nanomolar range affinity towards the target peptide. A mutation screen indicated that target-binding helices of increased homodimer stability and improved expression rates were preferred in the selection process. The crystal structure of the iM10/MITF-CC heterodimer (2.1Å) provided important structural insights and validated our design predictions. Importantly, iM10 did not only bind to the MITF coiled coil, but also to the markedly more stable HLHLZ domain of MITF. Characterizing the selected variants of the semi-rational library demonstrated the potential of the innovative bacterial selection approach.
Rotationally Invariant Image Representation for Viewing Direction Classification in Cryo-EM
Venue: Journal of structural biology

We introduce a new rotationally invariant viewing angle classification method for identifying, among a large number of cryo-EM projection images, similar views without prior knowledge of the molecule. Our rotationally invariant features are based on the bispectrum. Each image is denoised and compressed using steerable principal component analysis (PCA) such that rotating an image is equivalent to phase shifting the expansion coefficients. Thus we are able to extend the theory of bispectrum of 1D periodic signals to 2D images. The randomized PCA algorithm is then used to efficiently reduce the dimensionality of the bispectrum coefficients, enabling fast computation of the similarity between any pair of images. The nearest neighbors provide an initial classification of similar viewing angles. In this way, rotational alignment is only performed for images with their nearest neighbors. The initial nearest neighbor classification and alignment are further improved by a new classification method called vector diffusion maps. Our pipeline for viewing angle classification and alignment is experimentally shown to be faster and more accurate than reference-free alignment with rotationally invariant K-means clustering, MSA/MRA 2D classification, and their modern approximations.
Two and three dimensional characterization of Zucchini Yellow Mosaic Virus induced structural alterations in Cucurbita pepo L. plants
Venue: Journal of structural biology

Infection of plants by Zucchini Yellow Mosaic Virus (ZYMV) induces severe ultrastructural changes. The aim of this study was to investigate ultrastructural changes during ZYMV-infection in Cucurbita pepo L. plants on the two and three dimensional (2D and 3D) level and to correlate these changes with the spread of ZYMV throughout the plant by transmission electron microscopy (TEM) and image analysis. This study revealed that after inoculation of the cotyledons ZYMV moved into roots [3 days post inoculation (dpi)], then moved upwards into the stem and apical meristem (5 dpi), then into the first true leaf (7 dpi) and could finally be found in all plant parts (9 dpi). ZYMV-infected cells contained viral inclusion bodies in the form of cylindrical inclusions (CIs). These CIs occurred in four different forms throughout the cytosol of roots and leaves: scrolls and pinwheels when cut transversely and long tubular structures and bundles of filaments when cut longitudinally. 3D reconstruction of ZYMV-infected cells containing scrolls revealed that they form long tubes throughout the cytosol. The majority has a preferred orientation and an average length and width of 3 μm and 120 nm, respectively. Image analysis revealed an increased size of cells and vacuoles (107% and 447%, respectively) in younger ZYMV-infected leaves leading to a similar ratio of cytoplasm to vacuole (about 1:1) in older and younger ZYMV-infected leaves which indicates advanced cell growth in younger tissues. The collected data advances the current knowledge about ZYMV-induced ultrastructural changes in Cucurbita pepo.
Robust membrane detection based on tensor voting for electron tomography.
Venue: Journal of structural biology

Electron tomography enables three-dimensional (3D) visualization and analysis of the subcellular architecture at a resolution of a few nanometers. Segmentation of structural components present in 3D images (tomograms) is often necessary for their interpretation. However, it is severely hampered by a number of factors that are inherent to electron tomography (e.g. noise, low contrast, distortion). Thus, there is a need for new and improved computational methods to facilitate this challenging task. In this work, we present a new method for membrane segmentation that is based on anisotropic propagation of the local structural information using the tensor voting algorithm. The local structure at each voxel is then refined according to the information received from other voxels. Because voxels belonging to the same membrane have coherent structural information, the underlying global structure is strengthened. In this way, local information is easily integrated at a global scale to yield segmented structures. This method performs well under low signal-to-noise ratio typically found in tomograms of vitrified samples under cryo-tomography conditions and can bridge gaps present on membranes. The performance of the method is demonstrated by applications to tomograms of different biological samples and by quantitative comparison with standard template matching procedure.
Effect of sequence features on assembly of spider silk block copolymers.
Venue: Journal of structural biology

Bioengineered spider silk block copolymers were studied to understand the effect of protein chain length and sequence chemistry on the formation of secondary structure and materials assembly. Using a combination of in vitro protein design and assembly studies, we demonstrate that silk block copolymers possessing multiple repetitive units self-assemble into lamellar microstructures. Additionally, the study provides insights into the assembly behavior of spider silk block copolymers in concentrated salt solutions.
Single particle analysis integrated with microscopy: a high-throughput approach for reconstructing icosahedral particles.
Venue: Journal of structural biology

In cryo-electron microscopy and single particle analysis, data acquisition and image processing are generally carried out in sequential steps and computation of a three-dimensional reconstruction only begins once all the micrographs have been acquired. We are developing an integrated system for processing images of icosahedral particles during microscopy to provide reconstructed density maps in real-time at the highest possible resolution. The system is designed as a combination of pipelines to run in parallel on a computer cluster and analyzes micrographs as they are acquired, handling automatically all the processing steps from defocus estimation and particle picking to origin/orientation determination. An ab initio model is determined independently from the first micrographs collected, and new models are generated as more particles become available. As a proof of concept, we simulated data acquisition sessions using three sets of micrographs of good to excellent quality that were previously recorded from different icosahedral viruses. Results show that the processing of single micrographs can keep pace with an acquisition rate of about two images per minute. The reconstructed density map improves steadily during the image acquisition phase and its quality at the end of data collection is only moderately inferior to that obtained by expert users who processed semi-automatically all the micrographs after the acquisition. The current prototype demonstrates the advantages of integrating three-dimensional image processing with microscopy, which include an ability to monitor acquisition in terms of the final structure and to predict how much data and microscope resources are needed to achieve a desired resolution.
Motif Analyzer for protein 3D structures.
Venue: Journal of structural biology

The topology of the protein structure of all-β- or α/β-class is a special arrangement of β-strands within β-sheets (and α-helices surrounding β-sheets) and the order of them along the polypeptide chain. Structural motifs are a subset of strands and/or helices with widely spread topology. Structural motifs are used for classification of protein structure. Because of an increasing variety of known structures, an automatic tool for motif detection is needed. MotAn is an algorithmic detector of structural motifs in a given 3D protein structure. It detects β-hairpins, β-meanders, β-helices, Greek keys, interlocks, jellyrolls, β-α-β-motifs and β-α-β-helices. MotAn was tested on selected SCOP families and shown to be more sensitive detector than the PTGL and PROMOTIF programs. MotAn is available at http://mouse.belozersky.msu.ru/motan.
Crystal structure of the extracellular juxtamembrane region of Robo1.
Venue: Journal of structural biology

Robo receptors play pivotal roles in neurodevelopment, and their deregulation is implicated in several neuropathological conditions and cancers. To date, the mechanism of Robo activation and regulation remains obscure. Here we present the crystal structure of the juxtamembrane (JM) domains of human Robo1. The structure exhibits unexpectedly high backbone similarity to the netrin and RGM binding region of neogenin and DCC, which are functionally related receptors of Robo1. Comparison of these structures reveals a conserved surface that overlaps with a cluster of oncogenic and neuropathological mutations found in all Robo isoforms. The structure also reveals the intricate folding of the JM linker, which points to its role in Robo1 activation. Further experiments with cultured cells demonstrate that exposure or relief of the folded JM linker results in enhanced shedding of the Robo1 ectodomain.
Automated particle picking for low-contrast macromolecules in cryo-electron microscopy.
Venue: Journal of structural biology

Cryo-electron microscopy is an increasingly popular tool for studying the structure and dynamics of biological macromolecules at high resolution. A crucial step in automating single-particle reconstruction of a biological sample is the selection of particle images from a micrograph. We present a novel algorithm for selecting particle images in low-contrast conditions; it proves more effective than the human eye on close-to-focus micrographs, yielding improved or comparable resolution in reconstructions of two macromolecular complexes.
Cryo-EM analysis of the organization of BclA and BxpB in the Bacillus anthracis exosporium.
Venue: Journal of structural biology

Bacillus anthracis and other pathogenic Bacillus species form spores that are surrounded by an exosporium, a balloon-like layer that acts as the outer permeability barrier of the spore and contributes to spore survival and virulence. The exosporium consists of a hair-like nap and a paracrystalline basal layer. The filaments of the nap are comprised of trimers of the collagen-like glycoprotein BclA, while the basal layer contains approximately 20 different proteins. One of these proteins, BxpB, forms tight complexes with BclA and is required for attachment of essentially all BclA filaments to the basal layer. Another basal layer protein, ExsB, is required for the stable attachment of the exosporium to the spore. To determine the organization of BclA and BxpB within the exosporium, we used cryo-electron microscopy, cryo-sectioning and crystallographic analysis of negatively stained exosporium fragments to compare wildtype spores and mutant spores lacking BclA, BxpB or ExsB (ΔbclA, ΔbxpB and ΔexsB spores, respectively). The trimeric BclA filaments are attached to basal layer surface protrusions that appear to be trimers of BxpB. The protrusions interact with a crystalline layer of hexagonal subunits formed by other basal layer proteins. Although ΔbxpB spores retain the hexagonal subunits, the basal layer is not organized with crystalline order and lacks basal layer protrusions and most BclA filaments, indicating a central role for BxpB in exosporium organization.
Structural dynamics of V3 loop in a trimeric ambiance, a molecular dynamics study on gp120-CD4 trimeric mimic.
Venue: Journal of structural biology

Entry of HIV virus into the host cell is initiated by the interaction of its surface exposed gp120 protein with the cell surface CD4 receptor and a co-receptor that can be either CCR5 or CXCR4. The third variable region (V3 loop) of gp120 has an important role in co-receptor selection by gp120 and forms an epitope for neutralizing antibodies. In this work the dynamical behavior of the V3 loop in a trimeric environment has been investigated by generating an atomistic trimer model of gp120-CD4 complex and has been compared with the result of a monomeric gp120-CD4 complex. The main results coming from this work are that the three V3 loops belonging to the three subunits of the trimer display a different dynamical behavior in terms of its flexibility, spatial orientation, motion along the principal modes, conformations, solvent exposure and electrostatic potential distribution. We propose that the ability of the V3 loop to present, in the trimeric environment, simultaneous multiple alternative conformations that increase its capability of co-receptor recognition, is at least in part due to the effect of electrostatic potential generated by two subunits over the third one.
Structural diversity of a collagen-binding matrix protein from the byssus of blue mussels upon refolding.
Venue: Journal of structural biology

Blue mussels firmly adhere to a variety of different substrates by the byssus, an extracorporal structure consisting of several protein threads. These threads are mainly composed of fibrillar collagens called preCols which are embedded in a proteinaceous matrix. One of the two so far identified matrix proteins is the Proximal Thread Matrix Protein 1 (PTMP1). PTMP1 comprises two von Willebrand factor type A-like domains (A1 and A2) in a special arrangement. Here, we describe the refolding of recombinant PTMP1 from inclusion bodies. PTMP1 refolded into two distinct monomeric isoforms. Both isomers exhibited alternative intramolecular disulfide bonds. One of these isomers is thermodynamically favored and presumably represents the native form of PTMP1, while the other isoform is kinetically favored but is likely non-native. Oligomerization during refolding was influenced by, but not strictly dependent on disulfide formation. The conformational stability of PTMP1 indicates an influence of intramolecular disulfides on the native state, but not on unfolding intermediates.  PTMP1 exhibited a high thermal stability, dependent on the pH of the surrounding environment. Especially under acidic conditions the disulfide bonds were critically involved in thermal stability.
Web server for tilt-pair validation of single particle maps from electron cryomicroscopy.
Venue: Journal of structural biology

Three-dimensional structures of biological assemblies may be calculated from images of single particles obtained by electron cryomicroscopy. A key step is the correct determination of the orientation of the particle in individual image projections. A useful tool for validation of the quality of a 3D map and its consistency with images is tilt-pair analysis. In a successful tilt-pair test, the relative angle between orientations assigned to each image of a tilt-pair agrees with the known relative rotation angle of the microscope specimen holder during the experiment. To make the procedure easy to apply to the increasing number of single particle maps, we have developed software and a web server for tilt-pair analysis. The tilt-pair analysis program reports the overall agreement of the assigned orientations with the known tilt angle and axis of the experiment and the distribution of tilt transformations for individual particles recorded in a single image field. We illustrate application of the validation tool to several single particle specimens and describe how to interpret the scores.
A marker-free automatic alignment method based on scale-invariant features.
Venue: Journal of structural biology

In electron tomography, alignment accuracy is critical for high-resolution reconstruction. However, the automatic alignment of a tilt series without fiducial markers remains a challenge. Here, we propose a new alignment method based on Scale-Invariant Feature Transform (SIFT) for marker-free alignment. The method covers the detection and localization of interest points (features), feature matching, feature tracking and optimization of projection parameters. The proposed method implements a highly reliable matching strategy and tracking model to detect a huge number of feature tracks. Furthermore, an incremental bundle adjustment method is devised to tolerate noise data and ensure the accurate estimation of projection parameters. Our method was evaluated with a number of experimental data, and the results exhibit an improved alignment accuracy comparable with current fiducial marker alignment and subsequent higher resolution of tomography.
Decoration of silk fibroin by click chemistry for biomedical application.
Venue: Journal of structural biology

Silkfibroin (SF) has an excellent biocompatibility and its remarkable structure translates into exciting mechanical properties rendering this biomaterial particularly fascinating for biomedical application. To further boost the material's biological/preclinical impact, SF is decorated with biologics, typically by carbodiimide/N-hydroxysuccinimide coupling (EDC/NHS). For biomedical application, this chemistry challenges the product risk profile due to the formation of covalent aggregates, particularly when decoration is with biologics occurring naturally in humans as these aggregates may prime for autoimmunity. Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC; click chemistry) provides the necessary specificity to avoid such intermolecular, covalent aggregates. We present a blueprint outlining the necessary chemistry rendering SF compatible with CuAAC and with a particular focus on structural consequences. For that, the number of SF carboxyl groups (carboxyl-SF; required for EDC/NHS chemistry) or azido groups (azido-SF; required for click chemistry) was tailored by means of diazonium coupling of the SF tyrosine residues. Structural impact on SF and decorated SF was characterized by Fourier transform infrared spectroscopy (FTIR). The click chemistry yielded a better controlled product as compared to the EDC/NHS chemistry with no formation of inter- and intramolecular crosslinks as demonstrated for SF decorated with fluorescent model compounds or a biologic, fibroblast growth factor 2 (FGF2), respectively. In conclusion, SF can readily be translated into a scaffold compatible with click chemistry yielding decorated products with a better risk profile for biomedical application.