nils-herrmann/clean_scirepeval huggingface dataset:
C-Terminal acidic domain of ubiquitin-conjugating enzymes: a multi-functional conserved intrinsically disordered domain in family 3 of E2 enzymes.
Venue: Journal of structural biology

E2 ubiquitin-conjugating enzymes are key elements of the ubiquitin (Ub) pathway, since they influence processivity and topology of the Ub chain assembly and, as a consequence, the fate of the target substrates. E2s are multi-domain proteins, with accessory N-terminal or C-terminal domains that can contribute to the specificity for the cognate Ub-like molecules, or even the E3. In this context, the thorough structural characterization of E2 accessory domains is mandatory, in particular when they are associated to specific functions. We here provide, by computational and comparative studies, the first evidence of an acidic domain (AD) conserved in the E2 sub-family 3R. It is an intrinsically disordered domain, in which elements for Ub or E3 recognition are maintained. This conserved acidic domain (AD) shows propensity for α-helix structures (185-192 and 204-218) in the proximity of the sites for interaction with the Ub or the cognate E3. Moreover, our results also suggest that AD can explore conformations with tertiary contacts mainly driven by aromatic and hydrophobic interactions, in absence of its interaction partners. The globular states are likely to be regulated by multiple phosphorylation events, which can trigger conformational changes toward more extended conformations, as judged by MD simulations of the phospho-variants. The extended conformations, in turn, promote the accessibility of the interaction sites for Ub and the E3. We also trace a parallel between this new and natively unfolded structural motif for Ub-recognition and the natively folded ubiquitin associated domain (UBA) typical of family 1 of E2 enzymes, which includes Ubc1. In fact, according to our calculations, Ubc1 maps at the interface between the space of the natively unfolded and folded proteins, as well as it shares common features with the acidic domain of family 3 members.
Structure and function of the human sperm-specific isoform of protein kinase A (PKA) catalytic subunit Cα2.
Venue: Journal of structural biology

Protein kinase A (PKA) exists as several tissue-specific isoforms that through phosphorylation of serine and threonine residues of substrate proteins act as key regulators of a number of cellular processes. We here demonstrate that the human sperm-specific isoform of PKA named Cα2 is important for sperm motility and thus male fertility. Furthermore, we report on the first three-dimensional crystal structure of human apo Cα2 to 2.1 Å. Apo Cα2 displays an open conformation similar to the well-characterized apo structure of murine Cα1. The asymmetric unit contains two molecules and the core of the small lobe is rotated by almost 13° in the A molecule relative to the B molecule. In addition, a salt bridge between Lys72 and Glu91 was observed for Cα2 in the apo-form, a conformation previously found only in dimeric or ternary complexes of Cα1. Human Cα2 and Cα1 share primary structure with the exception of the amino acids at the N-terminus coded for by an alternative exon 1. The N-terminal glycine of Cα1 is myristoylated and this aliphatic chain anchors the N-terminus to an intramolecular hydrophobic pocket. Cα2 cannot be myristoylated and the crystal structure revealed that the equivalent hydrophobic pocket is unoccupied and exposed. Nuclear magnetic resonance (NMR) spectroscopy further demonstrated that detergents with hydrophobic moieties of different lengths can bind deep into this uncovered pocket. Our findings indicate that Cα2 through the hydrophobic pocket has the ability to bind intracellular targets in the sperm cell, which may modulate protein stability, activity and/or cellular localization.
Characterization of Escherichia coli nucleoids released by osmotic shock.
Venue: Journal of structural biology

Nucleoids were isolated by osmotic shock from Escherichia coli spheroplasts at relatively low salt concentrations and in the absence of detergents. Sucrose-protected cells, made osmotically sensitive by growth in the presence of ampicillin or by digestion with low lysozyme concentrations (50-5 μg/ml), were shocked by 100-fold dilution of the sucrose buffer. Liberated nucleoids stained with 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI), the dimeric cyanine dye TOTO-1, or fluorescent DNA-binding protein appeared as cloud-like structures, in the absence of phase contrast. Because UV-irradiation disrupted the DAPI-stained nucleoids within 5-10 s, they were imaged by time-lapse microscopy with exposure times less than 2 s. The volume of nucleoids isolated from ampicillin- or low-lysozyme spheroplasts and minimally exposed to UV (<2 s) was on average ∼42 μm(3). Lysozyme at concentrations above 1 μg/ml in the lysate compacted the nucleoids. Treatment with protease E or K (20-200 μg/ml) and sodium dodecyl sulfate (SDS; 0.001-0.01%) caused a twofold volume increase and showed a granular nucleoid at the earliest UV-exposure; the expansion could be reversed with 50 μM ethidium bromide, but not with chloroquine. While DNase (1 μg/ml) caused a rapid disruption of the nucleoids, RNase (0.1-400 μg/ml) had no effect. DAPI-stained nucleoids treated with protease, SDS or DNase consisted of granular substructures at the earliest exposure similar to UV-disrupted nucleoids obtained after prolonged (>4 s) UV irradiation. We interpret the measured volume in terms of a physical model of the nucleoid viewed as a branched DNA supercoil crosslinked by adhering proteins into a homogeneous network.
X-ray diffraction recording from single axonemes of eukaryotic flagella.
Venue: Journal of structural biology

We report the first X-ray diffraction patterns recorded from single axonemes of eukaryotic flagella with a diameter of only <0.2 μm, by using the technique of cryomicrodiffraction. A spermatozoon isolated from the testis of a fruit fly, Drosophila melanogaster, either intact or demembranated, was mounted straight in a glass capillary, quickly frozen and its 800-μm segment was irradiated end-on with intense synchrotron radiation X-ray microbeams (diameter, ~2 μm) at 74 K. Well-defined diffraction patterns were recorded, consisting of a large number of isolated reflection spots, extending up to 1/5 nm(-1). These reflections showed a tendency to peak every 20°, i.e., the patterns had features of an 18-fold rotational symmetry as expected from the 9-fold rotational symmetry of axonemal structure. This means that the axonemes remain untwisted, even after the manual mounting procedure. The diffraction patterns were compared with the results of model calculations based on a published electron micrograph of the Drosophila axoneme. The comparison provided information about the native state of axoneme, including estimates of axonemal diameter, interdoublet spacing, and masses of axonemal components relative to those of microtubules (e.g., radial spokes, dynein arms, and proteins associated with accessory singlet microtubules). When combined with the genetic resource of Drosophila, the technique presented here will serve as a powerful tool for studying the structure-function relationship of eukaryotic flagella in general.
Structure, composition, and mechanical properties of shark teeth.
Venue: Journal of structural biology

The teeth of two different shark species (Isurus oxyrinchus and Galeocerdo cuvier) and a geological fluoroapatite single crystal were structurally and chemically characterized. In contrast to dentin, enameloid showed sharp diffraction peaks which indicated a high crystallinity of the enameloid. The lattice parameters of enameloid were close to those of the geological fluoroapatite single crystal. The inorganic part of shark teeth consisted of fluoroapatite with a fluoride content in the enameloid of 3.1 wt.%, i.e., close to the fluoride content of the geological fluoroapatite single crystal (3.64 wt.%). Scanning electron micrographs showed that the crystals in enameloid were highly ordered with a special topological orientation (perpendicular towards the outside surface and parallel towards the center). By thermogravimetry, water, organic matrix, and biomineral in dentin and enameloid of both shark species were determined. Dentin had a higher content of water, organic matrix, and carbonate than enameloid but contained less fluoride. Nanoindentation and Vicker's microhardness tests showed that the enameloid of the shark teeth was approximately six times harder than the dentin. The hardness of shark teeth and human teeth was comparable, both for dentin and enamel/enameloid. In contrast, the geological fluoroapatite single crystal was much harder than both kinds of teeth due to the absence of an organic matrix. In summary, the different biological functions of the shark teeth ("tearing" for Isurus and "cutting" for Galeocerdo) are controlled by the different geometry and not by the chemical or crystallographic composition.
Exploring structural and thermodynamic stabilities of human prion protein pathogenic mutants D202N, E211Q and Q217R.
Venue: Journal of structural biology

The central event in the pathogenesis of prion protein (PrP) is a profound conformational change from its α-helical (PrP(C)) to its β-sheet-rich isoform (PrP(Sc)). Many single amino acid mutations of PrP are associated with familial prion diseases, such as D202N, E211Q, and Q217R mutations located at the third native α-helix of human PrP. In order to explore the underlying structural and dynamic effects of these mutations, we performed all-atom molecular dynamics (MD) simulations for the wild-type (WT) PrP and its mutants. The obtained results indicate that these amino acid substitutions have subtle effects on the protein structures, but show large changes of the overall electrostatic potential distributions. We can infer that the changes of PrP electrostatic surface due to the studied mutations may influence the intermolecular interactions during the aggregation process. In addition, the mutations also affect the thermodynamic stabilities of PrP.
Novel structural and functional insights into the MoxR family of AAA+ ATPases.
Venue: Journal of structural biology

The MoxR family of AAA+ ATPases is widespread among bacteria and archaea, although their cellular functions are not well characterized. Based on recent studies, MoxR ATPases are proposed to have chaperone-like function for the maturation of specific protein complexes or for the insertion of cofactors into proteins. MoxR proteins have been found to be important modulators of multiple stress response pathways in different organisms. For example, the respective MoxR proteins have been found to play important roles in the cell envelope stress response in Rhizobium leguminosarum, in the oxidative stress, acid stress, and heat stress responses in Francisella tularensis, in the acid stress and stringent responses in Escherichia coli, in viral tail formation in the crenarchaeal Acidianus two-tailed virus, and in the utilization of carbon monoxide as the sole carbon source by the Gram-negative chemolithoautotrophe Oligotropha carboxidovorans. Recent structural studies on the MoxR proteins from E. coli and Cytophaga hutchinsonii show the unique spatial arrangement of the αβα and all-α subdomains of the AAA+ domain in these proteins compared to the typical arrangement found in canonical AAA+ proteins such as HslU. The spatial organization of the subdomains in the AAA+ domain of MoxR proteins is similar to that found in the ATPase component of the magnesium chelatase complexes, possibly suggesting a similar mechanism of function. In this review, we provide an overview of the newly identified functions and the newly obtained structures of MoxR AAA+ ATPases.
Crystal structure of a mono- and diacylglycerol lipase from Malassezia globosa reveals a novel lid conformation and insights into the substrate specificity.
Venue: Journal of structural biology

Most lipases contain a lid domain to shield the hydrophobic binding site from the water environment. The lid, mostly in helical form, can undergo a conformational change to expose the active cleft during the interfacial activation. Here we report the crystal structures of Malassezia globosa LIP1 (SMG1) at 1.45 and 2.60 Å resolution in two crystal forms. The structures present SMG1 in its closed form, with a novel lid in loop conformation. SMG1 is one of the few members in the fungal lipase family that has been found to be strictly specific for mono- and diacylglycerol. To date, the mechanism for this substrate specificity remains largely unknown. To investigate the substrate binding properties, we built a model of SMG1 in open conformation. Based on this model, we found that the two bulky hydrophobic residues adjacent to the catalytic site and the N-terminal hinge region of the lid both may act as steric hindrances for triacylglycerols binding. These unique structural features of SMG1 will provide a better understanding on the substrate specificity of mono- and diacylglycerol lipases and a platform for further functional study of this enzyme.
Structure of Escherichia coli OmpF porin from lipidic mesophase.
Venue: Journal of structural biology

Outer membrane protein F, a major component of the Escherichia coli outer membrane, was crystallized for the first time in lipidic mesophase of monoolein in novel space groups, P1 and H32. Due to ease of its purification and crystallization OmpF can be used as a benchmark protein for establishing membrane protein crystallization in meso, as a "membrane lyzozyme". The packing of porin trimers in the crystals of space group H32 is similar to natural outer membranes, providing the first high-resolution insight into the close to native packing of OmpF. Surprisingly, interaction between trimers is mediated exclusively by lipids, without direct protein-protein contacts. Multiple ordered lipids are observed and many of them occupy identical positions independently of the space group, identifying preferential interaction sites of lipid acyl chains. Presence of ordered aliphatic chains close to a positively charged area on the porin surface suggests a position for a lipopolysaccharide binding site on the surface of the major E. coli porins.
Cell volume changes during apoptosis monitored in real time using digital holographic microscopy.
Venue: Journal of structural biology

Cellular volume changes play important roles in many processes associated with the normal cell activity, as well as various diseases. Consequently, there is a considerable need to accurately measure volumes of both individual cells and cell populations as a function of time. In this study, we have monitored cell volume changes in real time during apoptosis using digital holographic microscopy. Cell volume changes were deduced from the measured phase change of light transmitted through cells. Our digital holographic experiments showed that after exposure to 1 μM staurosporine for 4 h, the volumes of KB cells were reduced by ~50-60%, which is consistent with previous results obtained using electronic cell sizing and atomic force microscopy. In comparison with other techniques, digital holographic microscopy is advantageous because it employs noninvasive detection, has high time resolution, real time measurement capability, and the ability to simultaneously investigate time-dependent volume changes of both individual cells and cell populations.
Expanding into new markets--VCP/p97 in endocytosis and autophagy.
Venue: Journal of structural biology

The AAA-ATPase p97 (also called VCP for Valosin-containing protein) is essential for a number of cellular processes as diverse as ER-associated degradation, DNA damage response, and cell cycle control. Mechanistically, p97 cooperates with its cofactor Ufd1-Npl4 in these processes to segregate polyubiquitinated misfolded or regulatory client proteins from intracellular structures for subsequent degradation by the proteasome. Recent work now connects p97, independently of Ufd1-Npl4, to endosomal trafficking and autophagy. Interestingly, these pathways also deliver proteins for degradation, albeit by the lysosome. While monoubiquitination and alternative p97-cofactors, including UBXD1, have been associated with these activities, the underlying molecular mechanism(s) are still unclear or controversial. In this review, we aim to summarize the available data and discuss mechanistic models.
Crystal structure of the human spastin AAA domain.
Venue: Journal of structural biology

Hereditary spastic paraplegia (HSP) is a motor neuron disease caused by a progressive degeneration of the motor axons of the corticospinal tract. Point mutations or exon deletions in the microtubule-severing ATPase, spastin, are responsible for approximately 40% of cases of autosomal dominant HSP. Here, we report the 3.3 Å X-ray crystal structure of a hydrolysis-deficient mutant (E442Q) of the human spastin protein AAA domain. This structure is analyzed in the context of the existing Drosophila melanogaster spastin AAA domain structure and crystal structures of other closely related proteins in order to build a more unifying framework for understanding the structural features of this group of microtubule-severing ATPases.
Special issue on electron tomography.
Venue: Journal of structural biology

This special issue of the Journal of Structural Biology is devoted to electron tomography and is based on posters and presentations given at the 6th International Conference on Electron Tomography that took place May 2011, at the EMBL Heidelberg. The conference covered recent software and hardware developments as well as exciting applications exemplifying the diversity of biological fields in which electron tomography is considered as a central technique. Electron tomography and cryo-electron tomography in particular, became the method of choice for acquiring insights into the molecular organization and architecture of tissues, cells, organelles, and macromolecular assemblies, in situ. It is now considered to play a pivotal roll in answering fundamental questions in cellular and structural biology. The field of electron tomography has grown tremendously during the last decade as a result of improved software for 3D averaging and visualization of 3D images, the development of larger cameras and direct detection chips, as well as fully automated and high-throughput tilt-series collection systems for electron microscopes. Yet, there remains considerable room for improvement, especially in algorithms for classification of subtomograms and feature segmentation, as well as camera improvements, correlative light and electron microscopy and advanced specimen preparation techniques. Contributions to this special issue also address some of these issues. The application of cryo-electron tomography is of great interest since it provides the means for studying tissues and cells captured in a close-to-native state, but the methodology still requires a high level of expertise. Dose sensitivity and sample thickness remain major resolution limiting factor, however, improved detectors promise to revolutionize the current low-dose imaging capabilities. Preparation of frozen-hydrated specimens, especially the production of cryo-sections remains a bottleneck, but is of great
Automated tracing of filaments in 3D electron tomography reconstructions using Sculptor and Situs.
Venue: Journal of structural biology

The molecular graphics program Sculptor and the command-line suite Situs are software packages for the integration of biophysical data across spatial resolution scales. Herein, we provide an overview of recently developed tools relevant to cryo-electron tomography (cryo-ET), with an emphasis on functionality supported by Situs 2.7.1 and Sculptor 2.1.1. We describe a work flow for automatically segmenting filaments in cryo-ET maps including denoising, local normalization, feature detection, and tracing. Tomograms of cellular actin networks exhibit both cross-linked and bundled filament densities. Such filamentous regions in cryo-ET data sets can then be segmented using a stochastic template-based search, VolTrac. The approach combines a genetic algorithm and a bidirectional expansion with a tabu search strategy to localize and characterize filamentous regions. The automated filament segmentation by VolTrac compares well to a manual one performed by expert users, and it allows an efficient and reproducible analysis of large data sets. The software is free, open source, and can be used on Linux, Macintosh or Windows computers.
High-throughput subtomogram alignment and classification by Fourier space constrained fast volumetric matching.
Venue: Journal of structural biology

Cryo-electron tomography allows the visualization of macromolecular complexes in their cellular environments in close-to-live conditions. The nominal resolution of subtomograms can be significantly increased when individual subtomograms of the same kind are aligned and averaged. A vital step for such a procedure are algorithms that speedup subtomogram alignment and improve its accuracy to allow reference-free subtomogram classifications. Such methods will facilitate automation of tomography analysis and overall high throughput in the data processing. Building on previous work, here we propose a fast rotational alignment method that uses the Fourier equivalent form of a popular constrained correlation measure that considers missing wedge corrections and density variances in the subtomograms. The fast rotational search is based on 3D volumetric matching, which improves the rotational alignment accuracy in particular for highly distorted subtomograms with low SNR and tilt angle ranges in comparison to fast rotational matching of projected 2D spherical images. We further integrate our fast rotational alignment method in a reference-free iterative subtomogram classification scheme, and propose a local feature enhancement strategy in the classification process. As a proof of principle, we can demonstrate that the automatic method can successfully classify a large number of experimental subtomograms without the need of a reference structure.
Structural characterization of microcin E492 amyloid formation: Identification of the precursors.
Venue: Journal of structural biology

Microcin E492 is a low-molecular weight, channel-forming bacteriotoxin that generates amyloid structures. Using electron microscopy and image processing techniques several structural conformations can be observed. Prior to the conditions that induce amyloid formation and at its initial stage, microcin E492 molecules can be found in two main types of oligomers: a pentameric, pore-like structure consisting of globular monomers of ∼25Å diameter, and long filaments made up of stacked pentamers. The equilibrium between these structures depends on the properties of the solvent, because samples kept in methanol mainly show the pentameric structure. Amyloid induction in aqueous solvent reveals the presence, together with the above mentioned structures, of several amyloid structures such as flat and helical filaments. In addition, X-ray diffraction analysis demonstrated that the fibrils formed by microcin E492 presented cross-β structure, a distinctive property of amyloid fibrils. Based on the study of the observed structures we propose that microcin E492 has two conformations: a native one that assembles mainly into a pentameric structure, which functions as a pore, and an amyloid conformation which results in the formation of different types of amyloid filaments.