nils-herrmann/clean_scirepeval huggingface dataset:
TMaCS: a hybrid template matching and classification system for partially-automated particle selection.
Venue: Journal of structural biology

Selection of particle images from electron micrographs presents a bottleneck in determining the structures of macromolecular assemblies by single particle electron cryomicroscopy (cryo-EM). The problem is particularly important when an experimentalist wants to improve the resolution of a 3D map by increasing by tens or hundreds of thousands of images the size of the dataset used for calculating the map. Although several existing methods for automatic particle image selection work well for large protein complexes that produce high-contrast images, it is well known in the cryo-EM community that small complexes that give low-contrast images are often refractory to existing automated particle image selection schemes. Here we develop a method for partially-automated particle image selection when an initial 3D map of the protein under investigation is already available. Candidate particle images are selected from micrographs by template matching with template images derived from projections of the existing 3D map. The candidate particle images are then used to train a support vector machine, which classifies the candidates as particle images or non-particle images. In a final step in the analysis, the selected particle images are subjected to projection matching against the initial 3D map, with the correlation coefficient between the particle image and the best matching map projection used to assess the reliability of the particle image. We show that this approach is able to rapidly select particle images from micrographs of a rotary ATPase, a type of membrane protein complex involved in many aspects of biology.
Structure of glutaraldehyde cross-linked ryanodine receptor.
Venue: Journal of structural biology

The ryanodine receptor (RyR) family of calcium release channels plays a vital role in excitation-contraction coupling (ECC). Along with the dihydropyridine receptor (DHPR), calsequestrin, and several other smaller regulatory and adaptor proteins, RyRs form a large dynamic complex referred to as ECC machinery. Here we describe a simple cross-linking procedure that can be used to stabilize fragile components of the ECC machinery, for the purpose of structural elucidation by single particle cryo-electron microscopy (cryo-EM). As a model system, the complex of the FK506-binding protein (FKBP12) and RyR1 was used to test the cross-linking protocol. Glutaraldehyde fixation led to complete cross-linking of receptor-bound FKBP12 to RyR1, and also to extensive cross-linking of the four subunits comprising RyR to one another without compromising the RyR1 ultrastructure. FKBP12 cross-linked with RyR1 was visualized in 2D averages by single particle cryo-EM. Comparison of control RyR1 and cross-linked RyR1 3D reconstructions revealed minor conformational changes at the transmembrane assembly and at the cytoplasmic region. Intersubunit cross-linking enhanced [(3)H]ryanodine binding to RyR1. Based on our findings we propose that intersubunit cross-linking of RyR1 by glutaraldehyde induced RyR1 to adopt an open like conformation.
Crystal structure of Drosophila melanogaster tryptophan 2,3-dioxygenase reveals insights into substrate recognition and catalytic mechanism.
Venue: Journal of structural biology

Tryptophan 2,3-dioxygenase (TDO) catalyzes the oxidative cleavage of the indole ring of l-tryptophan to N-formylkynurenine in the kynurenine pathway, and is considered as a drug target for cancer immunotherapy. Here, we report the first crystal structure of a eukaryotic TDO from Drosophila melanogaster (DmTDO) in complex with heme at 2.7Å resolution. DmTDO consists of an N-terminal segment, a large domain and a small domain, and assumes a tetrameric architecture. Compared with prokaryotic TDOs, DmTDO contains two major insertion sequences: one forms part of the heme-binding site and the other forms a large portion of the small domain. The small domain which is unique to eukaryotic TDOs, interacts with the active site of an adjacent monomer and plays a role in the catalysis. Molecular modeling and dynamics simulation of DmTDO-heme-Trp suggest that like prokaryotic TDOs, DmTDO adopts an induced-fit mechanism to bind l-Trp; in particular, two conserved but flexible loops undergo conformational changes, converting the active site from an open conformation to a closed conformation. The functional roles of the key residues involved in recognition and binding of the heme and the substrate are verified by mutagenesis and kinetic studies. In addition, a modeling study of DmTDO in complex with the competitive inhibitor LM10 provides useful information for further inhibitor design. These findings reveal insights into the substrate recognition and the catalysis of DmTDO and possibly other eukaryotic TDOs and shed lights on the development of effective anti-TDO inhibitors.
Structure-guided studies of the SHP-1/JAK1 interaction provide new insights into phosphatase catalytic domain substrate recognition.
Venue: Journal of structural biology

SHP-1 (PTPN6) is a member of the SHP sub-family of protein tyrosine phosphatases and plays a critical role in the regulation of the JAK/STAT signaling pathway. Previous studies suggested that SHP-1 contains a PTP1B-like second phosphotyrosine pocket that allows for binding of tandem phosphotyrosine residues, such as those found in the activation loop of JAK kinases. To discover the structural nature of the interaction between SHP-1 and the JAK family member, JAK1, we determined the 1.8Å co-crystal structure of the SHP-1 catalytic domain and a JAK1-derived substrate peptide. This structure reveals electron density for only one bound phosphotyrosine residue. To investigate the role of the predicted second site pocket we determined the structures of SHP-1 in complex with phosphate and sulfate to 1.37Å and 1.7Å, respectively, and performed anomalous scattering experiments for a selenate-soaked crystal. These crystallographic data suggest that SHP-1 does not contain a PTP1B-like second site pocket. This conclusion is further supported by analysis of the relative dephosphorylation and binding affinities of mono- and tandem-phosphorylated peptide substrates. The crystal structures instead indicate that SHP-1 contains an extended C-terminal helix α2' incompatible with the predicted second phosphotyrosine binding site. This study suggests that SHP-1 defines a new category of PTP1B-like protein tyrosine phosphatases with a hindered second phosphotyrosine pocket.
Construction and organization of a BSL-3 cryo-electron microscopy laboratory at UTMB.
Venue: Journal of structural biology

A unique cryo-electron microscopy facility has been designed and constructed at the University of Texas Medical Branch (UTMB) to study the three-dimensional organization of viruses and bacteria classified as select agents at biological safety level (BSL)-3, and their interactions with host cells. A 200keV high-end cryo-electron microscope was installed inside a BSL-3 containment laboratory and standard operating procedures were developed and implemented to ensure its safe and efficient operation. We also developed a new microscope decontamination protocol based on chlorine dioxide gas with a continuous flow system, which allowed us to expand the facility capabilities to study bacterial agents including spore-forming species. The new unified protocol does not require agent-specific treatment in contrast to the previously used heat decontamination. To optimize the use of the cryo-electron microscope and to improve safety conditions, it can be remotely controlled from a room outside of containment, or through a computer network world-wide. Automated data collection is provided by using JADAS (single particle imaging) and SerialEM (tomography). The facility has successfully operated for more than a year without an incident and was certified as a select agent facility by the Centers for Disease Control.
Age-related nanostructural and nanomechanical changes of individual human cartilage aggrecan monomers and their glycosaminoglycan side chains.
Venue: Journal of structural biology

The nanostructure and nanomechanical properties of aggrecan monomers extracted and purified from human articular cartilage from donors of different ages (newborn, 29 and 38 year old) were directly visualized and quantified via atomic force microscopy (AFM)-based imaging and force spectroscopy. AFM imaging enabled direct comparison of full length monomers at different ages. The higher proportion of aggrecan fragments observed in adult versus newborn populations is consistent with the cumulative proteolysis of aggrecan known to occur in vivo. The decreased dimensions of adult full length aggrecan (including core protein and glycosaminoglycan (GAG) chain trace length, end-to-end distance and extension ratio) reflect altered aggrecan biosynthesis. The demonstrably shorter GAG chains observed in adult full length aggrecan monomers, compared to newborn monomers, also reflects markedly altered biosynthesis with age. Direct visualization of aggrecan subjected to chondroitinase and/or keratanase treatment revealed conformational properties of aggrecan monomers associated with chondroitin sulfate (CS) and keratan sulfate (KS) GAG chains. Furthermore, compressive stiffness of chemically end-attached layers of adult and newborn aggrecan was measured in various ionic strength aqueous solutions. Adult aggrecan was significantly weaker in compression than newborn aggrecan even at the same total GAG density and bath ionic strength, suggesting the importance of both electrostatic and non-electrostatic interactions in nanomechanical stiffness. These results provide molecular-level evidence of the effects of age on the conformational and nanomechanical properties of aggrecan, with direct implications for the effects of aggrecan nanostructure on the age-dependence of cartilage tissue biomechanical and osmotic properties.
Structural analysis of reactionary dentin formed in response to polymicrobial invasion.
Venue: Journal of structural biology

In response to microbial invasion of dentin odontoblasts secrete an altered calcified matrix termed reactionary dentin (Rd). 3D reconstruction of focused-ion-beam scanning electron microscopy (FIB-SEM) image slices revealed helical tubular structures in Rd that contrasted with regular cylindrical tubules characteristic of dentin from healthy teeth and affected so-called physiological dentin (Pd) lying exterior to Rd. This helical structure in Rd provided effective constriction of tubule lumen diameter that formed a barrier to bacterial advance towards the dental pulp. SEM of resin cast preparations revealed altered extension of odontoblast processes through Rd. The distribution of key mineral elements was studied by combination of 3D reconstruction of focused-ion-beam based X-ray microanalysis (FIB-EDS), laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS). There was a marked redistribution of calcium and phosphorous in Rd together with an increase of diffusely deposited magnesium compatible with the mineral deposition phase of synthesis of this altered matrix. Changes in tubule structure and mineral content characteristic of Rd are consistent with reduced hardness and lower elastic modulus reported for this matrix. Findings provide insight into the unique structure of Rd synthesised as a primary response to infection.
FASTDEF: fast defocus and astigmatism estimation for high-throughput transmission electron microscopy.
Venue: Journal of structural biology

In this work we present a fast and automated algorithm for estimating the contrast transfer function (CTF) of a transmission electron microscope. The approach is very suitable for High Throughput work because: (a) it does not require any initial defocus estimation, (b) it is almost an order of magnitude faster than existing approaches, (c) it opens the way to well-defined extensions to the estimation of higher order aberrations, at the same time that provides defocus and astigmatism estimations comparable in accuracy to well established methods, such as Xmipp and CTFFIND3 approaches. The new algorithm is based on obtaining the wrapped modulating phase of the power spectra density pattern by the use of a quadrature filter. This phase is further unwrapped in order to obtain the continuous and smooth absolute phase map; then a Zernike polynomial fitting is performed and the defocus and astigmatism parameters are determined. While the method does not require an initial estimation of the defocus parameters or any non-linear optimization procedure, these approaches can be used if further refinement is desired. Results of the CTF estimation method are presented for standard negative stained images, cryo-electron microscopy images in the absence of carbon support, as well as micrographs with only ice. Additionally, we have also tested the proposed method with micrographs acquired from tilted and untilted samples, obtaining good results. The algorithm is freely available as a part of the Xmipp package [http://xmipp.cnb.csic.es].
Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy.
Venue: Journal of structural biology

Correlative light and electron microscopy is an increasingly popular technique to study complex biological systems at various levels of resolution. Fluorescence microscopy can be employed to scan large areas to localize regions of interest which are then analyzed by electron microscopy to obtain morphological and structural information from a selected field of view at nm-scale resolution. Previously, an integrated approach to room temperature correlative microscopy was described. Combined use of light and electron microscopy within one instrument greatly simplifies sample handling, avoids cumbersome experimental overheads, simplifies navigation between the two modalities, and improves the success rate of image correlation. Here, an integrated approach for correlative microscopy under cryogenic conditions is presented. Its advantages over the room temperature approach include safeguarding the native hydrated state of the biological specimen, preservation of the fluorescence signal without risk of quenching due to heavy atom stains, and reduced photo bleaching. The potential of cryo integrated light and electron microscopy is demonstrated for the detection of viable bacteria, the study of in vitro polymerized microtubules, the localization of mitochondria in mouse embryonic fibroblasts, and for a search into virus-induced intracellular membrane modifications within mammalian cells.
X-ray structures of 4-chlorocatechol 1,2-dioxygenase adducts with substituted catechols: new perspectives in the molecular basis of intradiol ring cleaving dioxygenases specificity.
Venue: Journal of structural biology

The crystallographic structures of 4-chlorocatechol 1,2-dioxygenase (4-CCD) complexes with 3,5-dichlorocatechol, protocatechuate (3,4-dihydroxybenzoate), hydroxyquinol (benzen-1,2,4-triol) and pyrogallol (benzen-1,2,3-triol), which act as substrates or inhibitors of the enzyme, have been determined and analyzed. 4-CCD from the Gram-positive bacterium Rhodococcus opacus 1CP is a Fe(III) ion containing enzyme specialized in the aerobic biodegradation of chlorocatechols. The structures of the 4-CCD complexes show that the catechols bind the catalytic iron ion in a bidentate mode displacing Tyr169 and the benzoate ion (found in the native enzyme structure) from the metal coordination sphere, as found in other adducts of intradiol dioxygenases with substrates. The analysis of the present structures allowed to identify the residues selectively involved in recognition of the diverse substrates. Furthermore the structural comparison with the corresponding complexes of catechol 1,2-dioxygenase from the same Rhodococcus strain (Rho-1,2-CTD) highlights significant differences in the binding of the tested catechols to the active site of the enzyme, particularly in the orientation of the aromatic ring substituents. As an example the 3-substituted catechols are bound with the substituent oriented towards the external part of the 4-CCD active site cavity, whereas in the Rho-1,2-CTD complexes the 3-substituents were placed in the internal position. The present crystallographic study shed light on the mechanism that allows substrate recognition inside this class of high specific enzymes involved in the biodegradation of recalcitrant pollutants.
Cryo-electron tomography of the magnetotactic vibrio Magnetovibrio blakemorei: insights into the biomineralization of prismatic magnetosomes.
Venue: Journal of structural biology

We examined the structure and biomineralization of prismatic magnetosomes in the magnetotactic marine vibrio Magnetovibrio blakemorei strain MV-1 and a non-magnetotactic mutant derived from it, using a combination of cryo-electron tomography and freeze-fracture. The vesicles enveloping the Magnetovibrio magnetosomes were elongated and detached from the cell membrane. Magnetosome crystal formation appeared to be initiated at a nucleation site on the membrane inner surface. Interestingly, while scattered filaments were observed in the surrounding cytoplasm, their association with the magnetosome chains could not be unequivocally established. Our data suggest fundamental differences between prismatic and octahedral magnetosomes in their mechanisms of nucleation and crystal growth as well as in their structural relationships with the cytoplasm and plasma membrane.
Crystal structures of MBOgg1 in complex with two abasic DNA ligands.
Venue: Journal of structural biology

7,8-Dihydro-8-oxoguanine (8-oxoG) is one of the most common oxidative DNA lesions. 8-oxoguanine DNA glycosylases (Oggs) detect and excise 8-oxoG through a multiple-step process. To better understand the basis for estranged base recognition, we have solved the crystal structures of MBOgg1, the 8-oxoguanine DNA glycosylase of Thermoanaerobacter tengcongensis, in complex with DNA containing a tetrahydrofuranyl site (THF, a stable abasic site analog) paired with an estranged cytosine (MBOgg1/DNA(THF:C)) or thymine (MBOgg1/DNA(THF:T)). Different states of THF (extrahelical or intrahelical) are observed in the two complexes of the ASU of MBOgg1/DNA(THF:C) structure. Analyses of their different interaction modes reveal that variable contacts on the 5' region flanking the THF abasic site are correlated with the states of the THF. Comparison of MBOgg1/DNA(THF:T) with MBOgg1/DNA(THF:C) indicates that the non-preferred estranged T may affect MBOgg1's contacts with the 5' flank of the lesion strand. Furthermore, we identified a region in MBOgg1 that is rich in positive charges and interacts with the 5' region flanking the lesion. This region is conserved only in non-eukaryotic Oggs, and additional mutagenesis and biochemical assays reveal that it may contribute to the distinct estranged base specificities between eukaryotic and non-eukaryotic Oggs.
Dynamics in cryo EM reconstructions visualized with maximum-likelihood derived variance maps.
Venue: Journal of structural biology

CryoEM data capture the dynamic character associated with biological macromolecular assemblies by preserving the various conformations of the individual specimens at the moment of flash freezing. Regions of high variation in the data set are apparent in the image reconstruction due to the poor density that results from the lack of superposition of these regions. These observations are qualitative and, to date, only preliminary efforts have been made to quantitate the heterogeneity in the ensemble of particles that are individually imaged. We developed and tested a quantitative method for simultaneously computing a reconstruction of the particle and a map of the space-varying heterogeneity of the particle based on an entire data set. The method uses a maximum likelihood algorithm that explicitly takes into account the continuous variability from one instance to another instance of the particle. The result describes the heterogeneity of the particle as a variance to be plotted at every voxel of the reconstructed density. The test, employing time resolved data sets of virus maturation, not only recapitulated local variations obtained with difference map analysis, but revealed a remarkable time dependent reduction in the overall particle dynamics that was unobservable with classical methods of analysis.
Genesis of amorphous calcium carbonate containing alveolar plates in the ciliate Coleps hirtus (Ciliophora, Prostomatea).
Venue: Journal of structural biology

In the protist world, the ciliate Coleps hirtus (phylum Ciliophora, class Prostomatea) synthesizes a peculiar biomineralized test made of alveolar plates, structures located within alveolar vesicles at the cell cortex. Alveolar plates are arranged by overlapping like an armor and they are thought to protect and/or stiffen the cell. Although their morphology is species-specific and of complex architecture, so far almost nothing is known about their genesis, their structure and their elemental and mineral composition. We investigated the genesis of new alveolar plates after cell division and examined cells and isolated alveolar plates by electron microscopy, energy-dispersive X-ray spectroscopy, FTIR and X-ray diffraction. Our investigations revealed an organic mesh-like structure that guides the formation of new alveolar plates like a template and the role of vesicles transporting inorganic material. We further demonstrated that the inorganic part of the alveolar plates is composed out of amorphous calcium carbonate. For stabilization of the amorphous phase, the alveolar vesicles, the organic fraction and the element phosphorus may play a role.
Affinity grid-based cryo-EM of PKC binding to RACK1 on the ribosome.
Venue: Journal of structural biology

Affinity grids (AG) are specialized EM grids that bind macromolecular complexes containing tagged proteins to obtain maximum occupancy for structural analysis through single-particle EM. In this study, utilizing AG, we show that His-tagged activated PKC βII binds to the small ribosomal subunit (40S). We reconstructed a cryo-EM map which shows that PKC βII interacts with RACK1, a seven-bladed β-propeller protein present on the 40S and binds in two different regions close to blades 3 and 4 of RACK1. This study is a first step in understanding the molecular framework of PKC βII/RACK1 interaction and its role in translation.
Single versus dual-axis cryo-electron tomography of microtubules assembled in vitro: limits and perspectives.
Venue: Journal of structural biology

Single-axis cryo-electron tomography of vitrified specimens has become a method of choice to reconstruct in three dimensions macromolecular assemblies in their cellular context or prepared from purified components. Here, we asked how a dual-axis acquisition scheme would improve three-dimensional reconstructions of microtubules assembled in vitro. We show that in single-axis tomograms, microtubules oriented close to the perpendicular of the tilt axis display diminished contrast, and ultimately transform into sets of parallel lines oriented in the direction of the electron beam when observed in cross-section. Analysis of their three-dimensional Fourier transform indicates that this imaging artifact is due to a decrease in the angular sampling of their equatorial components. Although the second orthogonal series does not fully complement the first one at the specimen level due to increased radiation damage, it still allows elongated features oriented in any directions to be correctly reconstructed, which might be essential for highly heterogeneous specimens such as cells.