nils-herrmann/clean_scirepeval huggingface dataset:
Native structure of a retroviral envelope protein and its conformational change upon interaction with the target cell
Venue: Journal of structural biology

Enveloped viruses enter their host cells by membrane fusion. The process of attachment and fusion in retroviruses is mediated by a single viral envelope glycoprotein (Env). Conformational changes of Env in the course of fusion are a focus of intense studies. Here we provide further insight into the changes occurring in retroviral Env during its initial interaction with the cell, employing murine leukemia virus (MLV) as model system. We first determined the structure of both natively membrane anchored MLV Env and MLV Env tagged with YFP in the proline rich region (PRR) by electron cryo tomography (cET) and sub-volume averaging. At a resolution of ∼20 Å, native MLV Env presents as a hollow trimer (height ∼85 Å, diameter ∼120 Å) composed of step-shaped protomers. The major difference to the YFP-tagged protein was in regions outside of the central trimer. Next, we focused on elucidating the changes in MLV Env upon interaction with a host cell. Virus interaction with the plasma membrane occurred over a large surface and Env clustering on the binding site was observed. Sub-volume averaging did yield a low-resolution structure of Env interacting with the cell, which had lost its threefold symmetry and was elongated by ∼35 Å in comparison to the unbound protein. This indicates a major rearrangement of Env upon host cell binding. At the site of virus interaction, the otherwise clearly defined bilayer structure of the host cell plasma membrane was much less evident, indicative of integral membrane protein accumulation and/or a change in membrane lipid composition.
Structural insights into the centronuclear myopathy-associated functions of BIN1 and dynamin 2
Venue: Journal of structural biology

Centronuclear myopathies (CNMs) are genetic diseases whose symptoms are muscle weakness and atrophy (wasting) and centralised nuclei. Recent human genetic studies have isolated several groups of mutations. Among them, many are found in two interacting proteins essential to clathrin-mediated endocytosis, dynamin and the BIN-Amphiphysin-Rvs (BAR) protein BIN1/amphiphysin 2. In this review, by using structural and functional data from the study of endocytosis mainly, we discuss how the CNM mutations could affect the structure and the function of these ubiquitous proteins and cause the muscle-specific phenotype. The literature shows that both proteins are involved in the plasma membrane tubulation required for T-tubule biogenesis. However, this system also requires the regulation of the dynamin-mediated membrane fission, and the formation of a stable protein-scaffold to maintain the T-tubule structure. We discuss how the specific functions, isoforms and partners (myotubularin in particular) of these two proteins can lead to the establishment of muscle-specific features.
Three-dimensional CTF correction improves the resolution of electron tomograms.
Venue: Journal of structural biology

Correction of the contrast transfer function (CTF) of the microscope is a necessary step, in order to achieve high resolution from averaged electron microscopic images. Thereby, the CTF is first estimated and subsequently the electron micrograph is corrected, so that the negative oscillations of the CTF are equalized. Typically, the CTF correction is performed in 2D and the tilt-induced focus gradient is taken into account. Most often, the sample-thickness-induced focus gradient is ignored. Theoretical considerations, as well as implementation suggestions, for a 3D CTF correction that considers both gradients have been proposed before, although an implementation achieving a resolution improvement has been lacking, primarily due to computational reasons. Here, we present a comprehensive solution for a 3D CTF correction based on the Jensen-Kornberg scheme, which performs a slice-by-slice correction of the CTF within the tomographic reconstruction. We show that the computational requirements are comparable to those of 2D CTF correction. Using the examples of mitochondrial ribosomes and tobacco mosaic virus we demonstrate the improvement of the reconstruction quality with the 3D CTF correction, and the resolution gain on sub-tomogram averaging. More interestingly, for tomographic applications, the quality of the individual sub-tomograms before averaging increases significantly. We find that 3D CTF correction always produces equal or better results than 2D CTF correction.
Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?
Venue: Journal of structural biology

Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical β-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop.
Analogous biomineralization processes between the fossil coral Calceola sandalina (Rugosa, Devonian) and other Recent and fossil cnidarians.
Venue: Journal of structural biology

The current work represents a distinctive study about the biomineral properties of exceptionally good preserved skeletons of Calceola sandalina from the Middle Devonian of Couvin (Belgium), Smara (Morocco) and (Algeria) and their relation in the evolution of biomineralization of cnidarians. Structural and crystallographic analyses of the skeletons have been done by petrographic microscopy, electron scanning microscopy (SEM), atomic force microscopy (AFM), electron backscatter diffraction (EBSD), computer-integrated polarization microscopy (CIP) and electron microprobe analysis (EMPA). Calceola skeletons have many similarities with other cnidarians, mainly with other Palaeozoic corals as Syringoporicae: The microcrystals are composed of co-oriented nanocrystals that remind to mesocrystals, suggesting a biocrystallization process by particle attachment (CPA). The relationship between the nanocrystals and microcrystals suggest a growth mode similar to mineral bridges. A similar model was described for Syringoporicae corals (Tabulata) and it is similar to the coordinated-growth mode described in scleractinians and molluscs. Calceola skeletons show also a convergent structure with scleractinian forming Rapid Accretion Deposits (RAD), which share some structural and chemical properties. These evidences suggest analogous processes of biomineralization derived from a stem group of cnidarians. The results of this paper highlight the value of biomineralization studies in fossil organisms to understand the evolution of biomineralization mechanism through Phanerozoic.
Multiscale simulations of protein-facilitated membrane remodeling.
Venue: Journal of structural biology

Protein-facilitated shape and topology changes of cell membranes are crucial for many biological processes, such as cell division, protein trafficking, and cell signaling. However, the inherently multiscale nature of membrane remodeling presents a considerable challenge for understanding the mechanisms and physics that drive this process. To address this problem, a multiscale approach that makes use of a diverse set of computational and experimental techniques is required. The atomistic simulations provide high-resolution information on protein-membrane interactions. Experimental techniques, like electron microscopy, on the other hand, resolve high-order organization of proteins on the membrane. Coarse-grained (CG) and mesoscale computational techniques provide the intermediate link between the two scales and can give new insights into the underlying mechanisms. In this Review, we present the recent advances in multiscale computational approaches established in our group. We discuss various CG and mesoscale approaches in studying the protein-mediated large-scale membrane remodeling.
Physical interaction and assembly of Bacillus subtilis spore coat proteins CotE and CotZ studied by atomic force microscopy.
Venue: Journal of structural biology

The spore of Bacillus subtilis, a dormant type of cell, is surrounded by a complex multilayered protein structure known as the coat. It is composed of over 70 proteins and essential for the spore to withstand extreme environmental conditions and allow germination under favorable conditions. However, understanding how the properties of the coat arise from the interactions among all these proteins is an important challenge. Moreover, many specific protein-protein interactions among the coat proteins are crucial for coat assembly. In this study, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) was applied to investigate the interaction as a dynamic process between two morphogenetic coat proteins, CotE and CotZ. The unbinding force and kinetic parameters characterizing the interaction between CotE and CotZ were obtained. It is found that there is a strong affinity between CotE and CotZ. Furthermore, the assembly behaviors of CotE and CotZ, individually or in combination, were studied by AFM at solid-liquid interfaces. Our results revealed that CotE-CotZ assembly is dependent on their molar ratios and the interaction between CotE and CotZ involves in the CotE-CotZ assembly.
Axially aligned organic fibers and amorphous calcium phosphate form the claws of a terrestrial isopod (Crustacea).
Venue: Journal of structural biology

Skeletal elements that are exposed to heavy mechanical loads may provide important insights into the evolutionary solutions to mechanical challenges. We analyzed the microscopic architecture of dactylus claws in the woodlice Porcellio scaber and correlated these observations with analyses of the claws' mineral composition with energy dispersive X-ray spectrometry (EDX), electron energy loss spectroscopy (EELS) and selected area electron diffraction (SAED). Extraordinarily, amorphous calcium phosphate is the predominant mineral in the claw endocuticle. Unlike the strongly calcified exocuticle of the dactylus base, the claw exocuticle is devoid of mineral and is highly brominated. The architecture of the dactylus claw cuticle is drastically different from that of other parts of the exoskeleton. In contrast to the quasi-isotropic structure with chitin-protein fibers oriented in multiple directions, characteristic of the arthropod exoskeleton, the chitin-protein fibers and mineral components in the endocuticle of P. scaber claws are exclusively axially oriented. Taken together, these characteristics suggest that the claw cuticle is highly structurally anisotropic and fracture resistant and can be explained as adaptations to predominant axial loading of the thin, elongated claws. The nanoscale architecture of the isopod claw may inspire technological solutions in the design of durable machine elements subjected to heavy loading and wear.
Long shelf-life streptavidin support-films suitable for electron microscopy of biological macromolecules.
Venue: Journal of structural biology

We describe a rapid and convenient method of growing streptavidin (SA) monolayer crystals directly on holey-carbon EM grids. As expected, these SA monolayer crystals retain their biotin-binding function and crystalline order through a cycle of embedding in trehalose and, later, its removal. This fact allows one to prepare, and store for later use, EM grids on which SA monolayer crystals serve as an affinity substrate for preparing specimens of biological macromolecules. In addition, we report that coating the lipid-tail side of trehalose-embedded monolayer crystals with evaporated carbon appears to improve the consistency with which well-ordered, single crystals are observed to span over entire, 2μm holes of the support films. Randomly biotinylated 70S ribosomes are used as a test specimen to show that these support films can be used to obtain a high-resolution cryo-EM structure.
Defining the limits and reliability of rigid-body fitting in cryo-EM maps using multi-scale image pyramids.
Venue: Journal of structural biology

Cryo-electron microscopy provides fascinating structural insight into large macromolecular machines at increasing detail. Despite significant advances in the field, the resolution of the resulting three-dimensional images is still typically insufficient for de novo model building. To bridge the resolution gap and give an atomic interpretation to the data, high-resolution models are typically placed into the density as rigid bodies. Unfortunately, this is often done manually using graphics software, a subjective method that can lead to over-interpretation of the data. A more objective approach is to perform an exhaustive cross-correlation-based search to fit subunits into the density. Here we show, using five experimental ribosome maps ranging in resolution from 5.5 to 6.9Å, that cross-correlation-based fitting is capable of successfully fitting subunits correctly in the density for over 90% of the cases. Importantly, we provide indicators for the reliability and ambiguity of a fit, using the Fisher z-transformation and its associated confidence intervals, giving a formal approach to identify over-interpreted regions in the density. In addition, we quantify the resolution requirement for a successful fit as a function of the subunit size. For larger subunits the resolution of the data can be down-filtered to 20Å while still retaining an unambiguous fit. We leverage this information through the use of multi-scale image pyramids to accelerate the search up to 30-fold on CPUs and 40-fold on GPUs at a negligible loss in success rate. We implemented this approach in our rigid-body fitting software PowerFit, which can be freely downloaded from https://github.com/haddocking/powerfit.
Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging
Venue: Journal of structural biology

Cryo-electron tomography (cryoET) allows 3D structural information to be obtained from cells and other biological samples in their close-to-native state. In combination with subtomogram averaging, detailed structures of repeating features can be resolved. CryoET data is collected as a series of images of the sample from different tilt angles; this is performed by physically rotating the sample in the microscope between each image. The angles at which the images are collected, and the order in which they are collected, together are called the tilt-scheme. Here we describe a “dose-symmetric tilt-scheme” that begins at low tilt and then alternates between increasingly positive and negative tilts. This tilt-scheme maximizes the amount of high-resolution information maintained in the tomogram for subsequent subtomogram averaging, and may also be advantageous for other applications. We describe implementation of the tilt-scheme in combination with further data-collection refinements including setting thresholds on acceptable drift and improving focus accuracy. Requirements for microscope set-up are introduced, and a macro is provided which automates the application of the tilt-scheme within SerialEM.
Unraveling HIV protease flaps dynamics by Constant pH Molecular Dynamics simulations.
Venue: Journal of structural biology

The active site of HIV protease (HIV-PR) is covered by two flaps. These flaps are known to be essential for the catalytic activity of the HIV-PR, but their exact conformations at the different stages of the enzymatic pathway remain subject to debate. Understanding the correct functional dynamics of the flaps might aid the development of new HIV-PR inhibitors. It is known that, the HIV-PR catalytic efficiency is pH-dependent, likely due to the influence of processes such as charge transfer and protonation/deprotonation of ionizable residues. Several Molecular Dynamics (MD) simulations have reported information about the HIV-PR flaps. However, in MD simulations the protonation of a residue is fixed and thus it is not possible to study the correlation between conformation and protonation state. To address this shortcoming, this work attempts to capture, through Constant pH Molecular Dynamics (CpHMD), the conformations of the apo, substrate-bound and inhibitor-bound HIV-PR, which differ drastically in their flap arrangements. The results show that the HIV-PR flaps conformations are defined by the protonation of the catalytic residues Asp25/Asp25' and that these residues are sensitive to pH changes. This study suggests that the catalytic aspartates can modulate the opening of the active site and substrate binding.
Dynamo Catalogue: Geometrical tools and data management for particle picking in subtomogram averaging of cryo-electron tomograms.
Venue: Journal of structural biology

Cryo electron tomography allows macromolecular complexes within vitrified, intact, thin cells or sections thereof to be visualized, and structural analysis to be performed in situ by averaging over multiple copies of the same molecules. Image processing for subtomogram averaging is specific and cumbersome, due to the large amount of data and its three dimensional nature and anisotropic resolution. Here, we streamline data processing for subtomogram averaging by introducing an archiving system, Dynamo Catalogue. This system manages tomographic data from multiple tomograms and allows visual feedback during all processing steps, including particle picking, extraction, alignment and classification. The file structure of a processing project file structure includes logfiles of performed operations, and can be backed up and shared between users. Command line commands, database queries and a set of GUIs give the user versatile control over the process. Here, we introduce a set of geometric tools that streamline particle picking from simple (filaments, spheres, tubes, vesicles) and complex geometries (arbitrary 2D surfaces, rare instances on proteins with geometric restrictions, and 2D and 3D crystals). Advanced functionality, such as manual alignment and subboxing, is useful when initial templates are generated for alignment and for project customization. Dynamo Catalogue is part of the open source package Dynamo and includes tools to ensure format compatibility with the subtomogram averaging functionalities of other packages, such as Jsubtomo, PyTom, PEET, EMAN2, XMIPP and Relion.
A new method for vitrifying samples for cryoEM.
Venue: Journal of structural biology

Almost every aspect of cryo electron microscopy (cryoEM) has been automated over the last few decades. One of the challenges that remains to be addressed is the robust and reliable preparation of vitrified specimens of suitable ice thickness. We present results from a new device for preparing vitrified samples. The successful use of the device is coupled to a new "self-blotting" grid that we have developed to provide a method for spreading a sample to a thin film without the use of externally applied filter paper. This new approach has the advantage of using small amounts of protein material, resulting in large areas of ice of a well defined thickness containing evenly distributed single particles. We believe that these methods will in the future result in a system for vitrifying grids that is completely automated.
An algorithm for estimation and correction of anisotropic magnification distortion of cryo-EM images without need of pre-calibration.
Venue: Journal of structural biology

Anisotropic magnification distortion of TEM images (mainly the elliptic distortion) has been recently found as a potential resolution-limiting factor in single particle 3-D reconstruction. Elliptic distortions of ∼1-3% have been reported for multiple microscopes under low magnification settings (e.g., 18,000×), which significantly limited the achievable resolution of single particle 3-D reconstruction, especially for large particles. Here we report a generic algorithm that formulates the distortion correction problem as a generalized 2-D alignment task and estimates the distortion parameters directly from the particle images. Unlike the present pre-calibration methods, our computational method is applicable to all datasets collected at a broad range of magnifications using any microscope without need of additional experimental measurements. Moreover, the per-micrograph and/or per-particle level elliptic distortion estimation in our method could resolve potential distortion variations within a cryo-EM dataset, and further improve the 3-D reconstructions relative to constant-value correction by the pre-calibration methods. With successful applications to multiple datasets and cross-validation with the pre-calibration method, we have demonstrated the validity and robustness of our algorithm in estimating the distortion; correction of the elliptic distortion significantly improved the achievable resolutions by ∼1-3 folds and enabled 3-D reconstructions of multiple viral structures at 2.4-2.6Å resolutions. The resolution limits with elliptic distortion and the amounts of resolution improvements with distortion correction were found to strongly correlate with the product of the particle size and the amount of distortion, which can help assess if elliptic distortion is a major resolution limiting factor for single particle cryo-EM projects.
Structural snapshots of the catalytic cycle of the phosphodiesterase Autotaxin.
Venue: Journal of structural biology

Autotaxin (ATX) is a secreted phosphodiesterase that produces the signalling lipid lysophosphatidic acid (LPA). The bimetallic active site of ATX is structurally related to the alkaline phosphatase superfamily. Here, we present a new crystal structure of ATX in complex with orthovanadate (ATX-VO5), which binds the Oγ nucleophile of Thr209 and adopts a trigonal bipyramidal conformation, following the nucleophile attack onto the substrate. We have now a portfolio of ATX structures we discuss as intermediates of the catalytic mechanism: the new ATX-VO5 structure; a unique structure where the nucleophile Thr209 is phosphorylated (ATX-pThr). Comparing these to a complex with the LPA product (ATX-LPA) and with a complex with a phosphate ion (ATX-PO4), that represent the Michaelis complex of the reaction, we observe movements of Thr209, changes in the relative displacement of the zinc ions, and a water molecule that likely fulfils the second nucleophilic attack. We propose that ATX follows the associative two-step in-line displacement mechanism.
Membrane remodelling in bacteria.
Venue: Journal of structural biology

In bacteria the ability to remodel membrane underpins basic cell processes such as growth, and more sophisticated adaptations like inter-cell crosstalk, organelle specialisation, and pathogenesis. Here, selected examples of membrane remodelling in bacteria are presented and the diverse mechanisms for inducing membrane fission, fusion, and curvature discussed. Compared to eukaryotes, relatively few curvature-inducing proteins have been characterised so far. Whilst it is likely that many such proteins remain to be discovered, it also reflects the importance of alternative membrane remodelling strategies in bacteria where passive mechanisms for generating curvature are utilised.