nils-herrmann/clean_scirepeval huggingface dataset:
Segmentation by Classification: A Novel and Reliable Approach for Semi-Automatic Selection of HIV/SIV Envelope Spikes.
Venue: Journal of structural biology

We describe a semiautomated approach to segment Env spikes from the membrane envelope of Simian Immunodeficiency Virus visualized by cryoelectron tomography of frozen-hydrated specimens. Multivariate data analysis is applied to a large set of overlapping subvolumes extracted semiautomatically from the viral envelope and does not utilize a template of the target structure. The major manual step used in the method involves determination of six points that define an ellipsoid approximating the virion shape. The approach is robust to departures of the actual virion from this starting ellipsoid. A point cage of sufficient density is generated to ensure that any spike-like protein is identified multiple times. Subsequently translational alignment of class averages to a cylindrical reference on a curved surface separates subvolumes with spikes from those without. Spike containing subvolumes identified multiple times are removed by proximity analysis. Slightly different procedures segment spikes in the equatorial and the polar regions. Once all spikes are segmented, further alignment of class averages using separately the polar and spin angles produces recognizable spike images. Our approach localized 96% of the equatorial spikes and 85% of all spikes identified manually; it identifies a significant number of additional spikes missed by manual selection. Two types of spike shapes were segmented, one with near 3-fold symmetry resembling the conventional spike, the other had a T-shape resembling the spike structure obtained when antibodies such as PG9 bind to HIV Env. The approach should be applicable to segmentation of any protein spikes extending from a cellular or virion envelope.
Structural and functional investigation of AerF, a NADPH-dependent alkenal double bond reductase participating in the biosynthesis of Choi moiety of aeruginosin.
Venue: Journal of structural biology

The 2-carboxy-6-hydroxyoctahydroindole (Choi) moiety is an essential residue for the antithrombotic activities of aeruginosins, which are a class of cyanobacterial derived bioactive linear tetrapeptides. Biosynthetic pathway of Choi is still elusive. AerF was suggested to be involved in the biosynthesis of Choi, and can be assigned to the short-chain dehydrogenase/reductase (SDR) superfamily. However, both the exact role and the catalytic mechanism of AerF have not been elucidated. In this study, functional and mechanistic analyses of AerF from Microcystis aeruginosa were performed. Observation of enzymatic assay demonstrates that AerF is a NADPH-dependent alkenal double bond reductase that catalyzes the reduction of dihydro-4-hydroxyphenylpyruvate (H2HPP) to generate tetrahydro-4-hydroxyphenylpyruvate (H4HPP), which is the third step of the biosynthetic pathway from prephenate to Choi. Comparative structural analysis indicates that ligand binding-induced conformational change of AerF is different from that of the other SDR superfamily reductase using H2HPP as a substrate. Analyses of NADPH and substrate analogue binding sites combined with the results of mutagenesis analyses suggest that a particular serine residue mainly involves in the initiation of the proton transfer between the substrate and the residues of AerF, which is an uncommon feature in SDR superfamily reductase. Furthermore, based on the observations of structural and mutagenesis analyses, the catalytic mechanism of AerF is proposed and a proton transfer pathway in AerF is deduced.
Separating distinct structures of multiple macromolecular assemblies from cryo-EM projections.
Venue: Journal of structural biology

Single particle analysis for structure determination in cryo-electron microscopy is traditionally applied to samples purified to near homogeneity as current reconstruction algorithms are not designed to handle heterogeneous mixtures of structures from many distinct macromolecular complexes. We extend on long established methods and demonstrate that relating two-dimensional projection images by their common lines in a graphical framework is sufficient for partitioning distinct protein and multiprotein complexes within the same data set. The feasibility of this approach is first demonstrated on a large set of synthetic reprojections from 35 unique macromolecular structures spanning a mass range of hundreds to thousands of kilodaltons. We then apply our algorithm on cryo-EM data collected from a mixture of five protein complexes and use existing methods to solve multiple three-dimensional structures ab initio. Incorporating methods to sort single particle cryo-EM data from extremely heterogeneous mixtures will alleviate the need for stringent purification and pave the way toward investigation of samples containing many unique structures.
Tidy up cryo-EM sample grids with 3D printed tools.
Venue: Journal of structural biology

Cryo-EM technology has developed to the point of high-throughput structure determination of biological macromolecules embedded in vitreous ice. Nonetheless, challenging targets need extensive sample screening, often of many cryo-EM sample grids prepared under various conditions. We have designed and made tools for manipulating sample grids in storage cases. These tools are made of a plastic fiber using a wide-use 3D printer, a fused deposition modeling type, and polished under acetone gas. A grid case stacker organizes many frozen-hydrated cryo-EM grids and the stackers can be piled up inside a standard 50mL centrifuge tube. We have also introduced tools that facilitate handling of grid cases under liquid nitrogen and a stocker of the grid retainers contained in a CRYO ARM electron microscope. Blueprints of the tools named CryoGridTools are available from a GitHub site.
Conformational properties of l-fucose and the tetrasaccharide building block of the sulfated l-fucan from Lytechinus variegatus.
Venue: Journal of structural biology

Although the 3D structure of carbohydrates is known to contribute to their biological roles, conformational studies of sugars are challenging because their chains are flexible in solution and consequently the number of 3D structural restraints is limited. Here, we investigate the conformational properties of the tetrasaccharide building block of the Lytechinus variegatus sulfated fucan composed of the following structure [l-Fucp4(SO3-)-α(1-3)-l-Fucp2,4(SO3-)-α(1-3)-l-Fucp2(SO3-)-α(1-3)-l-Fucp2(SO3-)] and the composing monosaccharide unit Fucp, primarily by nuclear magnetic resonance (NMR) experiments performed at very low temperatures and using H2O as the solvent for the sugars rather than using the conventional deuterium oxide. By slowing down the fast chemical exchange rates and forcing the protonation of labile sites, we increased the number of through-space 1H-1H distances that could be measured by NMR spectroscopy. Following this strategy, additional conformational details of the tetrasaccharide and l-Fucp in solution were obtained. Computational molecular dynamics was performed to complement and validate the NMR-based measurements. A model of the NMR-restrained 3D structure is offered for the tetrasaccharide.
Lepidosaur ß-Keratin Chains With Four 34-Residue Repeats: Modelling Reveals A Potential Filament-Crosslinking Role.
Venue: Journal of structural biology

ß-keratin chains contain a characteristic and homologous 34-residue sequence, which is believed to adopt a twisted ß-sheet conformation that assembles in an antiparallel manner with a similar sheet in a second chain to form a ß-sandwich. These sandwiches are, in turn, related to one another by a left-handed four-fold screw axis to generate a helical structure that forms the core of the 3.4 nm diameter filaments observed by electron microscopy and deduced from X-ray fibre diffraction. Recently, it has been shown that one ß-keratin chain, with a molecular weight approximately twice that of the majority of ß-keratin chains, is conserved across the lepidosaurs (lizards, snakes and tuatara). Uniquely, it contains four 34-residue repeats. Although this chain is a minor component the observation that the entire chain shows a high degree of sequence conservation between species suggests an important structural/functional role in vivo. Modelling shows that only six families of structures are physically possible. In three of these the repeats exist within a single filament and might therefore act in a filament nucleation role. In the second three families the repeats exist in two, three or four filaments, implying that their function may be to act as an inter-filament crosslinker, thereby providing lateral reinforcement to the epidermal appendage. The favoured model is one in which the first two repeats form a β-sandwich in one filament and the second two repeats form a β-sandwich in a neighbouring filament. Links between alternating up- and down-pointing β-sheets would provide optimum connectivity.
How the central domain of dystrophin acts to bridge F-actin to sarcolemmal lipids.
Venue: Journal of structural biology

Dystrophin is a large intracellular protein that prevents sarcolemmal ruptures by providing a mechanical link between the intracellular actin cytoskeleton and the transmembrane dystroglycan complex. Dystrophin deficiency leads to the severe muscle wasting disease Duchenne Muscular Dystrophy and the milder allelic variant, Becker Muscular Dystrophy (DMD and BMD). Previous work has shown that concomitant interaction of the actin binding domain 2 (ABD2) comprising spectrin like repeats 11 to 15 (R11-15) of the central domain of dystrophin, with both actin and membrane lipids, can greatly increase membrane stiffness. Based on a combination of SAXS and SANS measurements, mass spectrometry analysis of cross-linked complexes and interactive low-resolution simulations, we explored in vitro the molecular properties of dystrophin that allow the formation of ABD2-F-actin and ABD2-membrane model complexes. In dystrophin we identified two subdomains interacting with F-actin, one located in R11 and a neighbouring region in R12 and another one in R15, while a single lipid binding domain was identified at the C-terminal end of R12. Relative orientations of the dystrophin central domain with F-actin and a membrane model were obtained from docking simulation under experimental constraints. SAXS-based models were then built for an extended central subdomain from R4 to R19, including ABD2. Overall results are compatible with a potential F-actin / dystrophin / membrane lipids ternary complex. Our description of this selected part of the dystrophin associated complex bridging muscle cell membrane and cytoskeleton opens the way to a better understanding of how cell muscle scaffolding is maintained through this essential protein.
Identification of a pH sensor in Influenza hemagglutinin using X-ray crystallography
Venue: Journal of Structural Biology

Abstract
 
 Hemagglutnin (HA) mediates entry of influenza virus through a series of conformational changes triggered by the low pH of the endosome. The residue or combination of residues acting as pH sensors has not yet been fully elucidated. In this work, we assay pH effects on the structure of H5 HA by soaking HA crystallized at pH 6.5 in a series of buffers with lower pH, mimicking the conditions of the endosome. We find that HA1-H38, which is conserved in Group 1 HA, undergoes a striking change in side chain conformation, which we attribute to its protonation and cation-cation repulsion with conserved HA1-H18. This work suggests that x-ray crystallography can be applied for studying small-scale pH-induced conformational changes providing valuable information on the location of pH sensors in HA. Importantly, the observed change in HA1-H38 conformation is further evidence that the pH-induced conformational changes of HA are the result of a series of protonation events to conserved and non-conserved pH sensors.
Asparagine-84, a regulatory allosteric site residue, helps maintain the quaternary structure of Campylobacter jejuni dihydrodipicolinate synthase.
Venue: Journal of structural biology

Dihydrodipicolinate synthase (DHDPS) from Campylobacter jejuni is a natively homotetrameric enzyme that catalyzes the first unique reaction of (S)-lysine biosynthesis and is feedback-regulated by lysine through binding to an allosteric site. High-resolution structures of the DHDPS-lysine complex have revealed significant insights into the binding events. One key asparagine residue, N84, makes hydrogen bonds with both the carboxyl and the α-amino group of the bound lysine. We generated two mutants, N84A and N84D, to study the effects of these changes on the allosteric site properties. However, under normal assay conditions, N84A displayed notably lower catalytic activity, and N84D showed no activity. Here we show that these mutations disrupt the quaternary structure of DHDPS in a concentration-dependent fashion, as demonstrated by size-exclusion chromatography, multi-angle light scattering, dynamic light scattering, small-angle X-ray scattering (SAXS) and high-resolution protein crystallography.
Dimerization of long Hibernation Promoting Factor from Staphylococcus aureus: structural analysis and biochemical characterization.
Venue: Journal of structural biology

Staphylococcus aureus hibernation promoting factor (SaHPF) is responsible for the formation of 100S ribosome dimers, which in turn help this pathogen to reduce energy spent under unfavorable conditions. Ribosome dimer formation strongly depends on the dimerization of the C-terminal domain of SaHPF (CTDSaHPF). In this study, we solved the crystal structure of CTDSaHPF at 1.6 Å resolution and obtained a precise arrangement of the dimer interface. Residues Phe160, Val162, Thr171, Ile173, Tyr175, Ile185 andThr187 in the dimer interface of SaHPF protein were mutated and the effects were analyzed for the formation of 100S disomes of ribosomes isolated from S. aureus. It was shown that substitution of any of single residues Phe160, Val162, Ile173, Tyr175 and Ile185 in the SaHPF homodimer interface abolished the ribosome dimerization in vitro.
Mineralized scale patterns on the cell periphery of the chrysophyte Mallomonas determined by comparative 3D Cryo-FIB SEM data processing.
Venue: Journal of structural biology

Unicellular protists can biomineralize spatially complex and functional shells. A typical cell of the photosynthetic synurophyte Mallomonas is covered by about 60-100 silica scales. Their geometric arrangement, the so-called scale case, mainly depends on the species and on the cell cycle. In this study, the scale case of the photosynthetic synurophyte Mallomonas was preserved in aqueous suspension using high-pressure freezing (HPF). From this specimen, a three-dimensional (3D) data set spanning a volume of about 25.6μm × 19.2μm × 4.2μm with a voxel size of 12.5nm × 12.5nm × 25.0nm was collected by Cryo-FIB SEM in 3 hours and 24 minutes. SEM imaging using In-lens SE detection allowed to clearly differentiate between mineralized, curved scales of less than 0.2μm thickness and organic cellular ultrastructure or vitrified ice. The three-dimensional spatial orientations and shapes of a minimum set of scales (N=13) were identified by visual inspection, and manually segmented. Manual and automated segmentation approaches were comparatively applied to one arbitrarily selected reference scale using the differences in grey level between scales and other constituents. Computational automated routines and principal component analysis of the experimentally extracted data created a realistic mathematical model based on the Fibonacci pattern theory. A complete in-silico scale case of Mallomonas was reconstructed showing an optimized scale coverage on the cell surface, similarly as it was observed experimentally. The minimum time requirements from harvesting the living cells to the final scale case determination by Cryo-FIB SEM and computational image processing are discussed.
Lateral A11 type tetramerization in lamins.
Venue: Journal of structural biology

The assembly of intermediate filaments (IFs) including nuclear lamins is driven by specific interactions of the elementary coiled-coil dimers in both lateral and longitudinal direction. The assembly mode A11 is dependent on lateral tetramerization of the second coiled-coil segment (coil1b) in antiparallel fashion. Recent cryo-electron microscopy studies pointed to 3.5 nm lamin filaments built from two antiparallel threads of longitudinally associated dimers but little molecular detail is available to date. Here we present the 2.6 Å resolution X-ray structure of a lamin A fragment including residues 65 to 222 which reveals the molecular basis of the A11 interaction. The crystal structure also indicates a continuous α-helical structure for the preceding linker L1 region. The middle part of the antiparallel tetramer reveals unique interactions due to the lamin-specific 42-residue insert in coil1b. At the same time, distinct characteristics of this insert provide for the preservation of common structural principles shared with lateral coil1b tetramers of vimentin and keratin K1/K10. In addition, structural analysis suggests that the A11 interaction in lamins is somewhat weaker than in cytoplasmic IFs, despite a 30% longer overlap. Establishing the structural detail of the A11 interaction across IF types is the first step towards a rational understanding of the IF assembly process which is indispensable for establishing the mechanism of disease-related mutations.
High-resolution structures of annexin A5 in a two-dimensional array.
Venue: Journal of structural biology

Annexins are soluble cytosolic proteins that bind to cell membranes. Annexin A5 self-assembles into a two-dimensional (2D) array and prevents cell rupture by attaching to damaged membranes. However, this process is not fully understood at the molecular level. In this study, we determined the crystal structures of annexin A5 with and without calcium (Ca2+) and confirmed the Ca2+-dependent outward motion of a tryptophan residue. Strikingly, the two structures exhibited the same crystal packing and 2D arrangement into a p3 lattice, which agrees well with the results of low-resolution structural imaging. High-resolution structures indicated that a three-fold interaction near the tryptophan residue is important for mediating the formation of the p3 lattice. A hypothesis on the promotion of p3 lattice formation by phosphatidyl serine (PS) is also suggested. This study provides molecular insight into how annexins modulate the physical properties of cell membranes as a function of Ca2+ concentration and the phospholipid composition of the membrane.
Modeling membrane proteins: the importance of cysteine amino-acids.
Venue: Journal of structural biology

Computational modeling of membrane proteins is critical to understand biochemical systems and to support chemical biology. In this work, we use a dataset of 448 non-redundant membrane protein chains to expose a "rule" that governs membrane protein structure: free cysteine thiols are not found accessible to oxidative compartments such as the extracellular space, but are rather involved in disulphide bridges. Taking as examples the 1018 three-dimensional models produced during the GPCR Dock 2008, 2010 and 2013 competitions and 390 models for a GPCR target in CASP13, we show that this rule was not accounted for by the modeling community. We thus highlight a new direction for model development that should lead to more accurate membrane protein models, especially in the loop domains.
Recognition of different base tetrads by RHAU: X-ray crystal structure of the G4 recognition motif bound to the 3'-end tetrad of a DNA G-quadruplex.
Venue: Journal of structural biology

G-quadruplexes (G4) are secondary structures of nucleic acids that can form in cells and have diverse biological functions. Several biologically important proteins interact with G-quadruplexes, of which RHAU - a helicase from the DEAH-box superfamily, was shown to bind and unwind G-quadruplexes efficiently. We report a X-ray co-crystal structure at 1.5 Å resolution of an N-terminal fragment of RHAU bound to the exposed tetrad of a parallel-stranded G-quadruplex. The RHAU peptide folds into an L-shaped α-helix, and binds to the G-quadruplex through π-stacking and electrostatic interactions. X-ray crystal structure of our complex identified key amino acid residues important for G-quadruplex-peptide binding interaction at the 3'-end G•G•G•G tetrad. Together with previous solution and crystal structures of RHAU bound to the 5'-end G•G•G•G and G•G•A•T tetrads, our crystal structure highlights the occurrence of a robust G-quadruplex recognition motif within RHAU that can adapt to different accessible tetrads.
Relationships between the structural and functional organization of the turtle cell nucleolus.
Venue: Journal of structural biology

The nucleolus is a multifunctional structure of the eukaryotic cell nucleus. However, its primary role is ribosome formation. Although the factors and mechanisms involved in ribogenesis are well conserved in eukaryotes, two types of nucleoli have been observed under the electron microscope: a tricompartmentalized nucleolus in amniotes and a bicompartmentalized nucleolus in other species. A recent study has also revealed that turtles, although belonging to amniotes, displayed a nucleolus with bipartite organization, suggesting that this reptile group may have carried out a reversion phenomenon during evolution. In this study, we examine in great detail the functional organization of the turtle nucleolus. In liver and spleen cells cultured in vitro, we confirm that the turtle nucleolus is mainly formed by two components: a fibrillar zone surrounded by a granular zone. We further show that the fibrillar zone includes densely-contrasted strands, which are positive after silver-stained Nucleolar Organizer Region (Ag-NOR) staining and DNA labelling. We also reveal that the dense strands condensed into a very compact mass within the fibrillar zone after a treatment with actinomycin D or 5,6-dichlorobenzimidazole riboside. Finally, by using pulse-chase experiments with BrUTP, three-dimensional image reconstructions of confocal optical sections, and electron microscopy analysis of ultrathin sections, we show that the topological and spatial dynamics of rRNA within the nucleolus extend from upstream binding factor (UBF)-positive sites in the fibrillar zone to the granular zone, without ever releasing the positive sites for the UBF. Together, these results seem to clearly indicate that the compartmentalization of the turtle nucleolus into two main components reflects a less orderly organization of ribosome formation.