nils-herrmann/clean_scirepeval huggingface dataset:
Ionic stress induces fusion of mitochondria to 3-D networks: An electron tomography study.
Venue: Journal of structural biology

Mitochondria are central organelles for energy supply of cells and play an important role in maintenance of ionic balance. Consequently mitochondria are highly sensitive to any kind of stress to which they mainly response by disturbance of respiration, ROS production and release of cytochrome c into the cytoplasm. Many of the physiological and molecular stress reactions of mitochondria are well known, yet there is a lack of information on corresponding stress induced structural changes. 3-D visualization of high-pressure frozen cells by FIB-SEM tomography and TEM tomography as used for the present investigation provide an excellent tool for studying structure related mitochondrial stress reactions. In the present study it is shown that mitochondria in the unicellular fresh-water algal model system Micrasterias as well as in the closely related aquatic higher plant Lemna fuse to local networks as a consequence of exposure to ionic stress induced by addition of KCl, NaCl and CoCl2. In dependence on concentration and duration of the treatment, fusion of mitochondria occurs either by formation of protuberances arising from the outer mitochondrial membrane, or by direct contact of the surface of elongated mitochondria. As our results show that respiration is maintained in both model systems during ionic stress and mitochondrial fusion, as well as formation of protuberances are reversible, we assume that mitochondrial fusion is a ubiquitous process that may help the cells to cope with stress. This may occur by interconnecting the respiratory chains of the individual mitochondria and by enhancing the buffer capacity against stress induced ionic imbalance.
Routine determination of ice thickness for cryo-EM grids.
Venue: Journal of structural biology

Recent advances in instrumentation and automation have made cryo-EM a popular method for producing near-atomic resolution structures of a variety of proteins and complexes. Sample preparation is still a limiting factor in collecting high quality data. Thickness of the vitreous ice in which the particles are embedded is one of the many variables that need to be optimized for collection of the highest quality data. Here we present two methods, using either an energy filter or scattering outside the objective aperture, to measure ice thickness for potentially every image collected. Unlike geometrical or tomographic methods, these can be implemented directly in the single particle collection workflow without interrupting or significantly slowing down data collection. We describe the methods as implemented into the Leginon/Appion data collection workflow, along with some examples from test cases. Routine monitoring of ice thickness should prove helpful for optimizing sample preparation, data collection, and data processing.
Processing apoferritin with the Appion pipeline.
Venue: Journal of structural biology

The 3DEM map challenge provided an opportunity to test different algorithms and workflows for processing single particle cryo-EM data. We were interested in testing whether we could use the standard Appion workflow with minimal manual intervention to achieve similar or better resolution than other challengers. Another question we were interested in testing was what the influence of particle sorting and elimination would be on the resolution and quality of 3D reconstructions. Since apoferritin is historically a challenging particle for single particle reconstruction and the authors of the original map challenge data used only a fraction of the particles present in the dataset, we focused on the apoferritin dataset for our entry. We submitted a 3.7 Å map from 25,844 particles and a 3.6 Å map from 53,334 particles and after assessment were among the best of the apoferritin maps that were submitted. Here we present the details of our reconstruction strategy and compare our strategy to that of another high-scoring apoferritin map. Altogether, our results suggest that for a relatively conformationally homogeneous particle like apoferritin, including as many particles as possible after elimination of junk leads to the highest resolution, and the choice of parameters for custom mask creation can lead to subtle but significant changes in the resolution of 3D reconstructions.
Characterization of inter-crystallite peptides in human enamel rods reveals contribution by the Y allele of amelogenin.
Venue: Journal of structural biology

Proteins of the inter-rod sheath and peptides within the narrow inter-crystallite space of the rod structure are considered largely responsible for visco-elastic and visco-plastic properties of enamel. The present study was designed to investigate putative peptides of the inter-crystallite space. Entities of 1-6 kDa extracted from enamel rods of erupted permanent teeth were analysed by mass spectrometry (MS) and shown to comprise N-terminal amelogenin (AMEL) peptides either containing or not containing exon 4 product. Other dominant entities consisted of an N-terminal peptide from ameloblastin (AMBN) and a series of the most hydrophobic peptides from serum albumin (ALBN). Amelogenin peptides encoded by the Y-chromosome allele were strongly detected in Enamel from male teeth. Location of N-terminal AMEL peptides as well as AMBN and ALBN, between apatite crystallites, was disclosed by immunogold scanning electron microscopy (SEM). Density plots confirmed the relative abundance of these products including exon 4+ AMEL peptides that have greater capacity for binding to hydroxyapatite. Hydrophilic X and Y peptides encoded in exon 4 differ only in substitution of non-polar isoleucine in Y for polar threonine in X with reduced disruption of the hydrophobic N-terminal structure in the Y form. Despite similarity of X and Y alleles of AMEL the non-coding region upstream from exon 4 shows significant variation with implications for segregation of processing of transcripts from exon 4. Detection of fragments from multiple additional proteins including keratins (KER), fetuin A (FETUA), proteinases and proteinase inhibitors, likely reflect biochemical events during enamel formation.
Detection of single alpha-helices in large protein sequence sets using hardware acceleration.
Venue: Journal of structural biology

Single alpha-helices (SAHs) are increasingly recognized as important structural and functional elements of proteins. Comprehensive identification of SAH segments in large protein datasets was largely hindered by the slow speed of the most restrictive prediction tool for their identification, FT_CHARGE on common hardware. We have previously implemented an FPGA-based version of this tool allowing fast analysis of a large number of sequences. Using this implementation, we have set up of a semi-automated pipeline capable of analyzing full UniProt releases in reasonable time and compiling monthly updates of a comprehensive database of SAH segments. Releases of this database, denoted CSAHDB, is available on the CSAHserver 2 website at csahserver.itk.ppke.hu. An overview of human SAH-containing sequences combined with a literature survey suggests specific roles of SAH segments in proteins involved in RNA-based regulation processes as well as cytoskeletal proteins, a number of which is also linked to the development and function of synapses.
PSF correction in soft X-ray tomography.
Venue: Journal of structural biology

In this article, we introduce a linear approximation of the forward model of soft X-ray tomography, such that the reconstruction is solvable by standard iterative schemes. This linear model takes into account the three-dimensional point spread function (PSF) of the optical system, which consequently enhances the reconstruction of data. The feasibility of the model is demonstrated on both simulated and experimental data, based on theoretically estimated and experimentally measured PSFs.
High resolution crystal structure of substrate-free human neprilysin.
Venue: Journal of structural biology

Neprilysin is a transmembrane M13 zinc metalloprotease responsible for the degradation of several biologically active peptides including insulin, enkephalin, substance P, bradykinin, endothelin-1, neurotensin and amyloid-β. The protein has received attention for its role in modulating blood pressure responses with its inhibition producing an antihypertensive response. To date, several inhibitor bound crystal structures of the human neprilysin extracellular domain have been determined, but, a structure free of bound inhibitor or substrate has yet to be reported. Here, we report the first crystal structure free of substrate or inhibitor for the extracellular catalytic domain of human neprilysin at 1.9 Å resolution. This structure will provide a reference point for comparisons to future inhibitor or substrate bound structures. The neprilysin structure also reveals that a closed protein conformation can be adopted in protein crystals absent of bound substrate or inhibitor.
Analytical symmetry detection in protein assemblies. II. Dihedral and cubic symmetries.
Venue: Journal of structural biology

Protein assemblies are often symmetric, as this organization has many advantages compared to individual proteins. Complex protein structures thus very often possess high-order symmetries. Detection and analysis of these symmetries has been a challenging problem and no efficient algorithms have been developed so far. This paper presents the extension of our cyclic symmetry detection method for higher-order symmetries with multiple symmetry axes. These include dihedral and cubic, i.e., tetrahedral, octahedral, and icosahedral, groups. Our method assesses the quality of a particular symmetry group and also determines all of its symmetry axes with a machine precision. The method comprises discrete and continuous optimization steps and is applicable to assemblies with multiple chains in the asymmetric subunits or to those with pseudo-symmetry. We implemented the method in C++ and exhaustively tested it on all 51,358 symmetric assemblies from the Protein Data Bank (PDB). It allowed us to study structural organization of symmetric assemblies solved by X-ray crystallography, and also to assess the symmetry annotation in the PDB. For example, in 1.6% of the cases we detected a higher symmetry group compared to the PDB annotation, and we also detected several cases with incorrect annotation. The method is available at http://team.inria.fr/nano-d/software/ananas. The graphical user interface of the method built for the SAMSON platform is available at http://samson-connect.net.
Structural and biochemical characterization of SpoIIIAF, a component of a sporulation-essential channel in Bacillus subtilis.
Venue: Journal of structural biology

Environmental stress factors initiate the developmental process of sporulation in some Gram-positive bacteria including Bacillus subtilis. Upon sporulation initiation the bacterial cell undergoes a series of morphological transformations that result in the creation of a single dormant spore. Early in sporulation, an asymmetric cell division produces a larger mother cell and smaller forespore. Next, the mother cell septal membrane engulfs the forespore, and an essential channel, the so-called feeding-tube apparatus, is formed. This assembled channel is thought to form a transenvelope secretion complex that crosses both mother cell and forespore membranes. At least nine proteins are essential for channel formation including SpoIIQ under forespore control and the eight SpoIIIA proteins (SpoIIIAA-AH) under mother cell control. Several of these proteins share similarity with components of Gram-negative bacterial secretion systems and the flagellum. Here we report the X-ray crystallographic structure of the soluble domain of SpoIIIAF to 2.7 Å resolution. Like the channel components SpoIIIAG and SpoIIIAH, SpoIIIAF adopts a conserved ring-building motif (RBM) fold found in proteins from numerous dual membrane secretion systems of distinct function. The SpoIIIAF RBM fold contains two unique features: an extended N-terminal helix, associated with multimerization, and an insertion at a loop region that can adopt two distinct conformations. The ability of the same primary sequence to adopt different secondary structure conformations is associated with protein regulation, suggesting a dual structural and regulatory role for the SpoIIIAF RBM. We further analyzed potential interaction interfaces by structure-guided mutagenesis in vivo. Collectively, our data provide new insight into the possible roles of SpoIIIAF within the secretion-like apparatus during sporulation.
Identification of a novel tetrameric structure for human apolipoprotein-D.
Venue: Journal of structural biology

Apolipoprotein-D is a 25 kDa glycosylated member of the lipocalin family that folds into an eight-stranded β-barrel with a single adjacent α-helix. Apolipoprotein-D specifically binds a range of small hydrophobic ligands such as progesterone and arachidonic acid and has an antioxidant function that is in part due to the reduction of peroxidised lipids by methionine-93. Therefore, apolipoprotein-D plays multiple roles throughout the body and is protective in Alzheimer's disease, where apolipoprotein-D overexpression reduces the amyloid-β burden in Alzheimer's disease mouse models. Oligomerisation is a common feature of lipocalins that can influence ligand binding. The native structure of apolipoprotein-D, however, has not been conclusively defined. Apolipoprotein-D is generally described as a monomeric protein, although it dimerises when reducing peroxidised lipids. Here, we investigated the native structure of apolipoprotein-D derived from plasma, breast cyst fluid (BCF) and cerebrospinal fluid. In plasma and cerebrospinal fluid, apolipoprotein-D was present in high-molecular weight complexes, potentially in association with lipoproteins. In contrast, apolipoprotein-D in BCF formed distinct oligomeric species. We assessed apolipoprotein-D oligomerisation using native apolipoprotein-D purified from BCF and a suite of complementary methods, including multi-angle laser light scattering, analytical ultracentrifugation and small-angle X-ray scattering. Our analyses showed that apolipoprotein-D predominantly forms a ∼95 to ∼100 kDa tetramer. Small-angle X-ray scattering analysis confirmed these findings and provided a structural model for apolipoprotein-D tetramer. These data indicate apolipoprotein-D rarely exists as a free monomer under physiological conditions and provide insights into novel native structures of apolipoprotein-D and into oligomerisation behaviour in the lipocalin family.
Solid-state NMR analysis of the sodium pump Krokinobacter rhodopsin 2 and its H30A mutant.
Venue: Journal of structural biology

Krokinobacter eikastus rhodopsin 2 (KR2) is a pentameric, light-driven ion pump, which selectively transports sodium or protons. The mechanism of ion selectivity and transfer is unknown. By using conventional as well as dynamic nuclear polarization (DNP)-enhanced solid-state NMR, we were able to analyse the retinal polyene chain between positions C10 and C15 as well as the Schiff base nitrogen in the KR2 resting state. In addition, 50% of the KR2 13C and 15N resonances could be assigned by multidimensional high-field solid-state NMR experiments. Assigned residues include part of the NDQ motif as well as sodium binding sites. Based on these data, the structural effects of the H30A mutation, which seems to shift the ion selectivity of KR2 primarily to Na+, could be analysed. Our data show that it causes long-range effects within the retinal binding pocket and at the extracellular Na+ binding site, which can be explained by perturbations of interactions across the protomer interfaces within the KR2 complex. This study is complemented by data from time-resolved optical spectroscopy.
Reductive power of the archaea right-handed coiled coil nanotube (RHCC-NT) and incorporation of mercury clusters inside protein cages.
Venue: Journal of structural biology

Coiled coils are well described as powerful oligomerization motifs and exhibit a large diversity of functions, including gene regulation, cell division, membrane fusion and drug extrusion. The archaea S-layer originated right-handed coiled coil -RHCC-NT- is characterized by extreme stability and is free of cysteine and histidine moieties. In the current study, we have followed a multidisciplinary approach to investigate the capacity of RHCC-NT to bind a variety of ionic complex metal ions. At the outside of the RHCC-NT, one mercury ion forms an electrostatic interaction with the S-methyl moiety of the single methionine residue present in each coil. We demonstrate that RHCC-NT is reducing and incorporating metallic mercury in the large-sized interior cavities which are lined up along the tetrameric channel.
cryoem-cloud-tools: A software platform to deploy and manage cryo-EM jobs in the cloud.
Venue: Journal of structural biology

Access to streamlined computational resources remains a significant bottleneck for new users of cryo-electron microscopy (cryo-EM). To address this, we have developed tools that will submit cryo-EM analysis routines and atomic model building jobs directly to Amazon Web Services (AWS) from a local computer or laptop. These new software tools ("cryoem-cloud-tools") have incorporated optimal data movement, security, and cost-saving strategies, giving novice users access to complex cryo-EM data processing pipelines. Integrating these tools into the RELION processing pipeline and graphical user interface we determined a 2.2 Å structure of ß-galactosidase in ∼55 h on AWS. We implemented a similar strategy to submit Rosetta atomic model building and refinement to AWS. These software tools dramatically reduce the barrier for entry of new users to cloud computing for cryo-EM and are freely available at cryoem-tools.cloud.
Biochemical characterization of the skeletal matrix of the massive coral, Porites australiensis - The saccharide moieties and their localization.
Venue: Journal of structural biology

To construct calcium carbonate skeletons of sophisticated architecture, scleractinian corals secrete an extracellular skeletal organic matrix (SOM) from aboral ectodermal cells. The SOM, which is composed of proteins, saccharides, and lipids, performs functions critical for skeleton formation. Even though polysaccharides constitute the major component of the SOM, its contribution to coral skeleton formation is poorly understood. To this end, we analyzed the SOM of the massive colonial coral, Porites australiensis, the skeleton of which has drawn great research interest because it records environmental conditions throughout the life of the colony. The coral skeleton was extensively cleaned, decalcified with acetic acid, and organic fractions were separated based on solubility. These fractions were analyzed using various techniques, including SDS-PAGE, FT-IR, in vitro crystallization, CHNS analysis, chromatography analysis of monosaccharide and enzyme-linked lectin assay (ELLA). We confirmed the acidic nature of SOM and the presence of sulphate, which is thought to initiate CaCO3 crystallization. In order to analyze glycan structures, we performed ELLA on the soluble SOM for the first time and found that it exhibits strong specificity to Datura stramonium lectin (DSL). Furthermore, using biotinylated DSL with anti-biotin antibody conjugated to nanogold, in situ localization of DSL-binding polysaccharides in the P. australiensis skeleton was performed. Signals were distributed on the surfaces of fiber-like crystals of the skeleton, suggesting that polysaccharides may modulate crystal shape. Our study emphasizes the importance of sugar moieties in biomineralization of scleractinian corals.
Spectral comparisons of mammalian cells and intact organelles by solid-state NMR.
Venue: Journal of structural biology

Whole-cell protein profiling, spatial localization, and quantification of activities such as gene transcription and protein translation are possible with modern biochemical and biophysical techniques. Yet, addressing questions of overall compositional changes within a cell - capturing the relative amounts of protein and ribosomal RNA levels and lipid content simultaneously - would require extractions and purifications with caveats due to isolation yields and detection methods. A holistic view of cellular composition would aid in the study of cellular composition and function. Here, solid state NMR is used to identify 13C NMR signatures for cellular organelles in HeLa cells without the use of any isotopic labeling. Comparisons are made with carbon spectra of subcellular assemblies including DNA, lipids, ribosomes, nuclei and mitochondria. Whole-cell comparisons are made with different mammalian cells lines, with red blood cells that lack nuclei and organelles, and with Gram-negative and Gram-positive bacteria. Furthermore, treatment of mammalian cells with cycloheximide, a commonly used protein synthesis inhibitor, revealed unanticipated changes consistent with a significant increase in protein glycosylation, obvious at the whole cell level. Thus, we demonstrate that solid-state NMR serves as a unique analytical tool to catalog and compare the ratios of distinct carbon types in cells and serves as a discovery tool to reveal the workings of inhibitors such as cycloheximide on whole-cell biochemistry.
Plasmin-driven fibrinolysis in a quasi-two-dimensional nanoscale fibrin matrix.
Venue: Journal of structural biology

Fibrin plays a fundamentally important role during hemostasis. To withstand the shear forces of blood flow and prevent embolisation, fibrin monomers form a three-dimensional polymer network that serves as an elastic scaffold for the blood clot. The complex spatial hierarchy of the fibrin meshwork, however, severely complicates the exploration of structural features, mechanical properties and molecular changes associated with the individual fibers of the clot. Here we developed a quasi-two-dimensional nanoscale fibrin matrix that enables the investigation of fibrin properties by topographical analysis using atomic force microscopy. The average thickness of the matrix was ∼50 nm, and structural features of component fibers were accessible. The matrix could be lysed with plasmin following rehydration. By following the topology of the matrix during lysis, we were able to uncover the molecular mechanisms of the process. Fibers became flexible but retained axial continuity for an extended time period, indicating that lateral interactions between protofibrils are disrupted first, but the axial interactions remain stable. Nearby fibers often fused into bundles, pointing at the presence of a cohesional force between them. Axial fiber fragmentation rapidly took place in the final step. Conceivably, the persisting axial integrity and cohesion of the fibrils assist to maintain global clot structure, to prevent microembolism, and to generate a high local plasmin concentration for the rapid, final axial fibril fragmentation. The nanoscale fibrin matrix developed and tested here provides a unique insight into the molecular mechanisms behind the structural and mechanical features of fibrin and its proteolytic degradation.