nils-herrmann/clean_scirepeval huggingface dataset:
An approach to automated acquisition of cryoEM images from lacey carbon grids.
Venue: Journal of structural biology

An approach to automated acquisition of cryoEM image data from lacey carbon grids using the Leginon program is described. Automated liquid nitrogen top up of the specimen holder dewar was used as a step towards full automation, without operator intervention during the course of data collection. During cryoEM studies of actin labelled with myosin V, we have found it necessary to work with lacey grids rather than Quantifoil or C-flat grids due to interaction of myosin V with the support film. Lacey grids have irregular holes of variable shape and size, in contrast to Quantifoil or C-flat grids which have a regular array of similar circular holes on each grid square. Other laboratories also prefer to work with grids with irregular holes for a variety of reasons. Therefore, it was necessary to develop a different strategy from normal Leginon usage for working with lacey grids for targeting holes for image acquisition and suitable areas for focussing prior to image acquisition. This approach was implemented by using the extensible framework provided by Leginon and by developing a new MSI application within that framework which includes a new Leginon node (for a novel method for finding focus targets).
Finite element modelling of cell wall properties for onion epidermis using a fibre-reinforced hyperelastic model.
Venue: Journal of structural biology

A combined finite element method and inverse modelling approach is used to model the mechanical deformation of onion epidermis. A fibre-reinforced hyperelastic composite material model considering the fibre distribution has been used to simulate the mechanical behaviour of samples under tension. The mechanical parameters of onion epidermis are determined using an inverse modelling approach. The simulated results show a good correlation with experimental observations.
Native architecture of the photosynthetic membrane from Rhodobacter veldkampii.
Venue: Journal of structural biology

The photosynthetic membrane in purple bacteria contains several pigment-protein complexes that assure light capture and establishment of the chemiosmotic gradient. The bioenergetic tasks of the photosynthetic membrane require the strong interaction between these various complexes. In the present work, we acquired the first images of the native outer membrane architecture and the supramolecular organization of the photosynthetic apparatus in vesicular chromatophores of Rhodobacter (Rb.) veldkampii. Mixed with LH2 (light-harvesting complex 2) rings, the PufX-containing LH1-RC (light-harvesting complex 1--reaction center) core complexes appear as C-shaped monomers, with random orientations in the photosynthetic membrane. Within the LH1 fence surrounding the RC, a remarkable gap that is probably occupied (or partially occupied) by PufX is visualized. Sequence alignment revealed that one specific region in PufX may be essential for PufX-induced core dimerization. In this region of ten amino acids in length all Rhodobacter species had five conserved amino acids, with the exception of Rb. veldkampii. Our findings provide direct evidence that the presence of PufX in Rb. veldkampii does not directly govern the dimerization of LH1-RC core complexes in the native membrane. It is indicated, furthermore, that the high membrane curvature of Rb. veldkampii chromatophores (Rb. veldkampii features equally small vesicular chromatophores alike Rb. sphaeroides) is not due to membrane bending induced by dimeric RC-LH1-PufX cores, as it has been proposed in Rb. sphaeroides.
Hysteretic swelling of wood at cellular scale probed by phase-contrast X-ray tomography.
Venue: Journal of structural biology

We investigated the three-dimensional, microscopic, dimensional changes of Picea abies (L. Karst) wood samples due to controlled steps of the ambient relative humidity. The study was performed at the wood cellular scale by high-resolution synchroton radiation phase-contrast X-ray tomographic microscopy (srPCXTM). Tomographic images were taken after the samples achieved moisture equilibrium at five adsorption and four desorption steps. For spruce latewood, swelling and shrinkage are found to be larger, more hysteretic and more homomorphic than for earlywood. Furthermore, while latewood undergoes similar strains in the transverse directions, earlywood radial strains are less than a third of the tangential strains. The less homomorphic and smaller swelling/shrinkage of earlywood in radial direction is found to be caused by the presence of rays.
Releasing of the chromophore from the drug delivery protein C-1027: a molecular dynamics simulations study.
Venue: Journal of structural biology

The aromatized chromophore (Chr) of C-1027 with selective DNA-cleaving ability, which is stabilized and delivered by the apoprotein (Apo) in vitro, is not released until the holoprotein (Apo+Chr) penetrates into the cultured cancer cells. As a drug delivery system, the holoprotein has gained much attention in clinical application. However, the Chr-releasing mechanism is ambiguous so far. In this paper, the releasing pathway is investigated using conventional molecular dynamics (MD), essential dynamics (ED), essential dynamics sampling and steered molecular dynamics (SMD) simulations. The results indicate that the releasing paths are related to the local motions of three loops: L3 (Val39-Gln42), L7 (Thr75-Thr79) and L9 (Asn97-Leu100). The major obstacles to Chr releasing come from steric hindrance, direct hydrogen bonds and hydrophobic interactions formed by the three loops, and Ser98 is an important residue in the releasing process. The most favorable direction of releasing is almost parallel to the connection between L7 and L3. Releasing from the direction, Chr only needs to break three hydrogen bonds from Ser98 and Pro76 and the weakest steric hindrance.
Structure and flexibility in cold-adapted iron superoxide dismutases: the case of the enzyme isolated from Pseudoalteromonas haloplanktis.
Venue: Journal of structural biology

Superoxide dismutases (SODs) are metalloenzymes catalysing the dismutation of superoxide anion radicals into molecular oxygen and hydrogen peroxide. Here, we present the crystal structure of a cold-adapted Fe-SOD from the Antarctic eubacterium Pseudoalteromonas haloplanktis (PhSOD), and that of its complex with sodium azide. The structures were compared with those of the corresponding homologues having a high sequence identity with PhSOD, such as the mesophilic SOD from Escherichia coli (EcSOD) or Pseudomonas ovalis, and the psychrophilic SOD from Aliivibrio salmonicida (AsSOD). These enzymes shared a large structural similarity, such as a conserved tertiary structure and arrangement of the two monomers, an almost identical total number of inter- and intramolecular hydrogen bonds and salt bridges. However, the two cold-adapted SODs showed an increased flexibility of the active site residues with respect to their mesophilic homologues. Structural information was combined with a characterisation of the chemical and thermal stability performed by CD and fluorescence measurements. Despite of its psychrophilic origin, the denaturation temperature of PhSOD was comparable with that of the mesophilic EcSOD, whereas AsSOD showed a lower denaturation temperature. On the contrary, the values of the denaturant concentration at the transition midpoint were in line with the psychrophilic/mesophilic origin of the proteins. These data provide additional support to the hypothesis that cold-adapted enzymes achieve efficient catalysis at low temperature, by increasing the flexibility of their active site; moreover, our results underline how fine structural modifications can alter enzyme flexibility and/or stability without compromising the overall structure of typical rigid enzymes, such as SODs.
Arrangement of trichokeratin intermediate filaments and matrix in the cortex of Merino wool.
Venue: Journal of structural biology

Tomograms of transverse sections of Merino wool fibers obtained from fleeces differing in fiber curvature were reconstructed from image series collected using a 300kV transmission electron microscope. Trichokeratin intermediate filaments (IFs) from the ortho-, para- and mesocortices were modeled from the tomograms. IFs were predominantly arranged in left-handed concentric helices with the relative angle of IFs increasing progressively from the center to the periphery of orthocortex macrofibrils. The median increase in IF angle between adjacent IFs between the center and periphery was 2.5°. The length of one turn of the helical path of an IF was calculated to be approximately 1μm for an IF tilted at 30° and positioned 100nm from the macrofibril center. With the exception of one paracortex macrofibril that weakly resembled an orthocortex macrofibril, all para- and mesocortex macrofibrils modeled had a parallel arrangement of the IFs, with a more ordered arrangement found in the mesocortex. Within the limited sample set, there appeared to be no significant relationship between IF angle and fiber curvature. We examined the matrix/IF ratio (in the form of proportion of matrix to one IF, calculated from IF center-to-center distance and IF diameter) for 28 macrofibrils used for modeling. The proportion of matrix was significantly different in the different cortex cell types, with paracortex having the most (0.61), orthocortex having the least (0.42), and mesocortex being intermediate (0.54). Fibers of different crimp type (high, medium or low crimp) were not significantly different from each other with respect to matrix proportion.
Reprint of: Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film.
Venue: Journal of structural biology

Direct observation of subcellular structures and their characterization is essential for understanding their physiological functions. To observe them in open environment, we have developed an inverted scanning electron microscope with a detachable, open-culture dish, capable of 8 nm resolution, and combined with a fluorescence microscope quasi-simultaneously observing the same area from the top. For scanning electron microscopy from the bottom, a silicon nitride film window in the base of the dish maintains a vacuum between electron gun and open sample dish while allowing electrons to pass through. Electrons are backscattered from the sample and captured by a detector under the dish. Cells cultured on the open dish can be externally manipulated under optical microscopy, fixed, and observed using scanning electron microscopy. Once fine structures have been revealed by scanning electron microscopy, their component proteins may be identified by comparison with separately prepared fluorescence-labeled optical microscopic images of the candidate proteins, with their heavy-metal-labeled or stained ASEM images. Furthermore, cell nuclei in a tissue block stained with platinum-blue were successfully observed without thin-sectioning, which suggests the applicability of this inverted scanning electron microscope to cancer diagnosis. This microscope visualizes mesoscopic-scale structures, and is also applicable to non-bioscience fields including polymer chemistry.
Maximum likelihood based classification of electron tomographic data.
Venue: Journal of structural biology

Classification and averaging of sub-tomograms can improve the fidelity and resolution of structures obtained by electron tomography. Here we present a three-dimensional (3D) maximum likelihood algorithm--MLTOMO--which is characterized by integrating 3D alignment and classification into a single, unified processing step. The novelty of our approach lies in the way we calculate the probability of observing an individual sub-tomogram for a given reference structure. We assume that the reference structure is affected by a 'compound wedge', resulting from the summation of many individual missing wedges in distinct orientations. The distance metric underlying our probability calculations effectively down-weights Fourier components that are observed less frequently. Simulations demonstrate that MLTOMO clearly outperforms the 'constrained correlation' approach and has advantages over existing approaches in cases where the sub-tomograms adopt preferred orientations. Application of our approach to cryo-electron tomographic data of ice-embedded thermosomes revealed distinct conformations that are in good agreement with results obtained by previous single particle studies.
The interstitial crystal-nucleating sheet in molluscan Haliotis rufescens shell: a bio-polymeric composite.
Venue: Journal of structural biology

The interstitial green sheets in abalone shell nacre are shown to be bifacially differentiated trilaminate polymeric complexes, with glycoprotein layers sandwiching a central core containing chitin. They share some common feature with the organic matrix layers between the aragonite tablets in the nacre and the periostracum, and show similarities to the myostracum. Thus, although the green sheet is reported to be unique to the abalone shell, it represents an interesting model for the study of molluscan shell biomineralization processes. Indeed, during shell formation, prismatic and spherulitic aragonite precedes and follows the deposition of the interstitial green polymeric composite sheets, and there is evidence to suggest that these sheets demark the interruption of nacre synthesis and serve to nucleate the resumption of calcium carbonate crystal growth. The green polymeric interstitial sheet purified from the abalone shell was investigated by spectroscopic and imaging techniques: FTIR, confocal microscopy, scanning and transmission electron microscopy, and by pyrolysis combined with GC-MS. Structural and compositional differences are observed between the surfaces of the two sides of the interstitial polymeric composite sheets. Moreover, comparative crystallization experiments on the green sheet sides also reveal asymmetry with respect to the nucleation of calcium carbonate. These findings suggest that these bifacially differentiated interstitial composites may play an active role in the mineral assembly processes, with one of the surfaces acting as a crystal nucleator.
Zernike phase contrast cryo-electron tomography of sodium-driven flagellar hook-basal bodies from Vibrio alginolyticus.
Venue: Journal of structural biology

Vibrio alginolyticus use flagella to swim. A flagellum consists of a filament, hook and basal body. The basal body is made up of a rod and several ring structures. This study investigates the structure of the T ring which is a unique component of the V. alginolyticus sodium ion-driven flagellar basal body. Using Zernike phase contrast (ZPC) cryo-electron tomography, we compared the 3D structures of purified hook-basal bodies (HBB) from a wild-type strain (KK148) and a deletion mutant lacking MotX and MotY (TH3), which are thought to form the T ring. ZPC images of HBBs had highly improved signal-to-noise ratio compared to conventional phase contrast images. We observed the outline of the HBBs from strains KK148 and TH3, and the TH3 mutant was missing its T ring. In the wild-type strain, the T ring was beneath the LP ring and seemed to form a ring shape with diameter of 32 nm.
Nanoscale morphology of Type I collagen is altered in the Brtl mouse model of Osteogenesis Imperfecta.
Venue: Journal of structural biology

Bone has a complex hierarchical structure that has evolved to serve structural and metabolic roles in the body. Due to the complexity of bone structure and the number of diseases which affect the ultrastructural constituents of bone, it is important to develop quantitative methods to assess bone nanoscale properties. Autosomal dominant Osteogenesis Imperfecta results predominantly from glycine substitutions (80%) and splice site mutations (20%) in the genes encoding the α1 or α2 chains of Type I collagen. Genotype-phenotype correlations using over 830 collagen mutations have revealed that lethal mutations are located in regions crucial for collagen-ligand binding in the matrix. However, few of these correlations have been extended to collagen structure in bone. Here, an atomic force microscopy-based approach was used to image and quantitatively analyze the D-periodic spacing of Type I collagen fibrils in femora from heterozygous (Brtl/+) mice (α1(I)G349C), compared to wild type (WT) littermates. This disease system has a well-defined change in the col1α1 allele, leading to a well characterized alteration in collagen protein structure, which are directly related to altered Type I collagen nanoscale morphology, as measured by the D-periodic spacing. In Brtl/+ bone, the D-periodic spacing shows significantly greater variability on average and along the length of the bone compared to WT, although the average spacing was unchanged. Brtl/+ bone also had a significant difference in the population distribution of collagen D-period spacings. These changes may be due to the mutant collagen structure, or to the heterogeneity of collagen monomers in the Brtl/+ matrix. These observations at the nanoscale level provide insight into the structural basis for changes present in bone composition, geometry and mechanical integrity in Brtl/+ bones. Further studies are necessary to link these morphological observations to nanoscale mechanical integrity.
On the evolutionary origins of "Fold Space Continuity": a study of topological convergence and divergence in mixed alpha-beta domains.
Venue: Journal of structural biology

Existing protein structure classifications group proteins by overall structural similarity at the highest level and by evolutionary relationships at the lowest level, deriving higher-level groups by pairwise structure comparison. For this to be successful requires that large changes in structure are relatively rare in evolution and that proteins with no detectable evolutionary relationship do not converge on similar global chain conformations since this creates conflicts between structural and evolutionary consistency. Analysis of global structural changes using core topological descriptions for 4261 domains from classes C and D of the SCOP database and new measures of topological distance and consistency of classification showed that the topological consistency of SCOP folds is highly variable with some folds having no consistent description and significant overlaps between groups including some members of separate folds with identical topological descriptions. Topological clustering shows that including sufficient indels to allow family members to be joined would also require joining several distinct folds. We conclude that evolutionary changes in the global topology of protein domains are the root cause of many difficulties for present approaches to structure classification using pairwise comparison. As a resolution we propose that a purely structural classification should be created using an approach similar to that adopted by the Gene Ontology in which proteins are assigned labels describing structure.
New trends in protein expression.
Venue: Journal of structural biology

In order to benefit from the wealth of genomics data and to make new discoveries in the protein world, genes need to be converted to proteins that, in turn, must be characterized at the molecular level. This has galvanized researchers from across a broad range of biological and biomedical disciplines to study the form and function of thousands of proteins. This task can be achieved by production of stable and pure proteins for their biophysical and biochemical analysis and/or for their 3D structure determination. The broad hierarchy of structures in biological systems necessitates integration of a wide range of high and low-resolution structural determination techniques. These include X-ray and neutron crystallography, NMR, electron microscopy and tomography, small angle X-ray and neutron scattering, mass spectroscopy and advanced light microscopy techniques. Common to all these techniques is the requirement for highly pure protein samples. Consequently, high throughput protein expression and purification, as well as more versatile expression systems and techniques, are now in even greater demand to support the pressing requirements of the research pipeline. However, protein expression is not only a technology, but also an art. Each protein is unique and thus requires special and often tedious treatment. Therefore, the expression of high levels of stable and functional proteins remains a bottleneck in many scientific endeavors, including the determination of structures in a high-throughput fashion or the screening for novel active compounds in modern drug discovery. Recently, numerous developments have substantially improved the procedures for production of soluble and active proteins in heterologous expression systems. These include parallel construct design, new and more robust expression systems, modifications to the expression constructs, introduction of new and/or improved prokaryotic and eukaryotic expression systems, and the development of improved cell-free protein synthesis systems. This has driven technological developments striving for miniaturization and automation, and methods to characterize proteins prior to structural characterization, in order to achieve a better understanding of how to design constructs generating proteins more amenable to structural characterization. While many of these developments have gained momentum from the various ‘‘structural genomics/proteomics initiatives”, they are now being applied to study the form and function of proteins by scientists from a broad range of biological and biomedical disciplines. We present here a collection of papers in a special issue entitled ‘‘New Trends in Protein Expression”. Our goal was to gather papers concentrating on current developments of the methods and tools that are being used in the production of proteins for structural analysis. In assembling the articles appearing in this volume of the Journal of Structural Biology, an attempt has been made to focus on those principles, strategies, and technologies that currently
Crystal structure of MexZ, a key repressor responsible for antibiotic resistance in Pseudomonas aeruginosa.
Venue: Journal of structural biology

Pseudomonas aeruginosa is responsible for around 10% of all hospital-acquired infections and the single most important pathogen of cystic fibrosis lungs. P. aeruginosa has high intrinsic and acquired antibiotic resistance, due to the extrusion of antibiotics by multidrug efflux pumps. The gene regulator MexZ controls the expression of mexXY, the efflux pump responsible for resistance to many drugs that are used for treating CF patients. MexZ is shown to be the most frequently mutated gene in P. aeruginosa isolated from CF patient lungs, confirming its importance in multidrug resistance. Here we present the crystal structure of MexZ at 2.9Å. Combining the structural information with biochemical data on key mutants identified, we provide an explanation for the structural and functional consequences of these mutants. This work provides a framework for further characterisation of MexZ in order to fully understand its regulation and induction.