nils-herrmann/clean_scirepeval huggingface dataset:
Mechanical properties of modern calcite- (Mergerlia truncata) and phosphate-shelled brachiopods (Discradisca stella and Lingula anatina) determined by nanoindentation.
Venue: Journal of structural biology

We measured distribution patterns of hardness and elastic modulus by nanoindentation on shells of the rhynchonelliform brachiopod Mergerlia truncata and the linguliform brachiopods Discradisca stella and Lingula anatina. The rhynchonelliformea produce calcitic shells while the linguliformea produce chitinophosphatic shells. Dorsal and ventral valves, commissure and hinge of the calcitic shell of M. truncata show different nanohardness values (from 2.3 to 4.6 GPa) and E-modulus (from 52 to 76 GPa). The hardness of the biocalcite is always increased compared to inorganic calcite. We attribute the effects to different amounts of inter- and intracrystalline organic matrix. Profiles parallel to the radius of curvature of the valves cutting through the different layers of shell material surprisingly show quite uniform values of nanohardness and modulus of elasticity. Nanoindentation tests on the chitinophosphatic brachiopods D. stella and L. anatina reflect the hierarchical structure composed of laminae with varying degree of mineralization. As a result of the two-phase composite of biopolymer nanofibrils reinforced with Ca-phosphate nanoparticles, nanohardness, and E-modulus correlate almost linearly from (H=0.25 GPa, E=2.5 GPa) to (H=2.5 GPa, E=50 GPa). The mineral provides stiffness and hardness, the biopolymer provides flexibility; and the composite provides fracture toughness. Gradients in the degree of mineralization reduce potential stress concentrations at the interface between stiff mineralized and soft non-mineralized laminae. For the epibenthic chitinophosphatic D. stella the lamination is also present but less pronounced than for the infaunal L. anatina, and the overall distribution of material strength in the cross-sectional profile shows a maximum in the center and a decrease towards the inner and outer shell margins (modulus of elasticity from 30 to 12 GPa, hardness from 1.7 to 0.5 GPa). Accordingly, the two epibenthic forms, calcitic M. truncata and chitinophosphatic D. stella display fairly bulky (homogeneous) nanomechanical properties of their shell materials, while the burrowing infaunal L. anatina is distinctively laminated. The strongly mineralized laminae, which provide the strength to the shell, are also brittle, but keeping them as thin as possible, allows some bending flexibility. This flexibility is not required for the epibenthic life style.
Alport syndrome mutations in type IV tropocollagen alter molecular structure and nanomechanical properties.
Venue: Journal of structural biology

Alport Syndrome is a genetic disease characterized by breakdown of the glomerular basement membrane (GBM) around blood vessels in the kidney, leading to kidney failure in most patients. It is the second most inherited kidney disease in the US, and many other symptoms are associated with the disease, including hearing loss and ocular lesions. Here we probe the molecular level structure-property relationships of this disease using a bottom-up computational materiomics approach implemented through large-scale molecular dynamics simulation. Since the GBM is under constant mechanical loading due to blood flow, changes in mechanical properties due to amino acid mutations may be critical in the symptomatic GBM breakdown seen in Alport Syndrome patients. Through full-atomistic simulations in explicit solvent, the effects of single-residue glycine substitution mutations of varying clinical severity are studied in short segments of type IV tropocollagen molecules. The segments with physiological amino acid sequences are equilibrated and then subjected to tensile loading. Major changes are observed at the single molecule level of the mutated sequence, including a bent shape of the structures after equilibration (with the kink located at the mutation site) and a significant alteration of the molecules' stress-strain responses and stiffnesses. These results suggest that localized structural changes at amino acid level induce severe alterations of the molecular properties. Our study opens a new approach in pursuing a bottom-up multi-scale analysis of this disease.
Dynamics of silica cell wall morphogenesis in the diatom Cyclotella cryptica: substructure formation and the role of microfilaments.
Venue: Journal of structural biology

Diatoms are unicellular algae that make cell walls out of silica with highly ornate features on the nano- to microscale. The complexity and variety of diatom cell wall structures exceeds those possible with synthetic materials chemistry approaches. Understanding the design and assembly processes involved in diatom silicification should provide insight into patterning on the unicellular level, and information for biomimetic approaches for materials synthesis. In this report we examine the formation of distinct cell wall structures (valves and girdle bands) in the diatom Cyclotella cryptica by high resolution imaging using SEM, AFM, and fluorescence microscopy. Special attention was paid to imaging structural intermediates, which provided insight into the underlying design and assembly principles involved. Distinct stages in valve formation were identified, indicating a transition from a fractally organized structure to a dynamic pathway-dependent process. Substructures in the valves appeared to be pre-positioned prior to complete silicification, suggesting that organics responsible for these structures were pre-assembled and put in place. Microtubules and microfilamentous actin play significant roles in the positioning process, and actin is also important in the pathway-dependent expansion of the front of silicification. Our results indicate that even though all silica structures in C. cryptica are made of assemblies of nanoparticulate silica, control of meso- and microscale structure occurs on a higher order. It is apparent that diatoms integrate bottom up and top down control and synthesis mechanisms to form the diversity of structures possible.
NADPH oxidase activator p67(phox) behaves in solution as a multidomain protein with semi-flexible linkers.
Venue: Journal of structural biology

The NADPH oxidase complex is involved in the destruction of phagocytosed pathogens through the production of reactive oxygen species. This activatable complex consists of a membranous heterodimeric flavocytochrome b, a small G-protein Rac1/Rac2 and cytosolic factors, p47(phox), p67(phox) and p40(phox). p67(phox), due to its modular structure, is the NADPH oxidase component for which global structure information is most scarce despite its mandatory role in activation and its central position in the whole complex organization. Indeed, p67(phox) is the only factor establishing interaction with all others. In this study, we report the SAXS analysis of p67(phox). Our data reveals that p67(phox) behaves as a multidomain protein with semi-flexible linkers. On the one hand, it appears to be a very elongated molecule with its various domains organized as beads on a string. Linkers are predicted to be partially or mainly unstructured and features of our experimental data do point towards inter-domain flexibility. On the other hand, our work also suggests that the protein is not as extended as unstructured linkers could allow, thereby implying the existence of intra-molecular interactions within p67(phox). We suggest that the dual character of p67(phox) conformation in solution is central to ensure the numerous interactions to be accommodated.
Histomorphometric analysis of adult articular calcified cartilage zone.
Venue: Journal of structural biology

The aim of this study was to carry out a histomorphometric analysis of calcified cartilage zone (CCZ) and its interfaces between hyaline cartilage and subchondral bone. The study used 40 donated normal human femoral condyles, from which paraffin-embedded sections were prepared after fixation and decalcification. The histomorphology of the CCZ were qualitatively and quantitatively observed by staining, scanning electron microscopy (SEM) and three-dimensional (3D) reconstruction. The hyaline cartilage and CCZ were stained red with Safranin-O, and the subchondral bone was stained blue with Fast green. CCZ was stained black after von Kossa staining. The hyaline cartilage was interlocked tightly in the manner of "ravine-engomphosis" by the CCZ. The surface roughnesses of tidemark and cement line were 1.14+/-0.04 and 1.99+/-0.38. The maximum, minimum and mean thicknesses of CCZ were 277.12+/-8.6, 9.83+/-6.72 and 104.162+/-0.87 microm, respectively. The cell density of CCZ (51.25+/-21.26 cells/mm(2)) was significantly lower than that of the hyaline cartilage (152.54+/-35.77 cells/mm(2)) (P<0.05). The subchondral bone was anchored tightly in the manner of a "comb-anchor" by the CCZ in our 3D reconstruction model. Thus, we discovered two junctional interfaces of CCZ using different histomorphometric methods. The upper interface of CCZ is a "ravine-engomphosis" shape, while its lower interface is a "comb-anchor" shape.
Localization and orientation of the gamma-tubulin small complex components using protein tags as labels for single particle EM.
Venue: Journal of structural biology

Gamma-Tubulin Small Complex (gamma-TuSC) is the universally-conserved complex in eukaryotes that contains the microtubule (MT) nucleating protein: gamma-tubulin. gamma-TuSC is a heterotetramer with two copies of gamma-tubulin and one copy each of Spc98p and Spc97p. Previously, the structure of gamma-TuSC was determined by single particle electron microscopy (EM) at 25A resolution. gamma-TuSC is Y-shaped with a single flexible arm that could be the key to regulating MT nucleation. EM gold labeling revealed the locations of gamma-tubulin at the top of the Y. In vivo Fluorescence Resonance Energy Transfer (FRET) suggested the relative orientations of Spc98p and Spc97p but did not distinguish which large subunit formed the flexible arm. Here, using fluorescent proteins as covalently attached tags, we used class averages and 3-D random conical tilt reconstructions to confirm the in vivo FRET results, clearly demonstrating that the Spc98p/97p C-termini interact directly with gamma-tubulin. Most significantly we have determined that the flexible arm belongs to Spc98p and our data also suggests that the N-termini of Spc98p and Spc97p are crossed. More generally, our results confirm that despite their small size, covalently-attached fluorescent proteins perform well as subunit labels in single particle EM.
Distinct in situ structures of the Borrelia flagellar motor.
Venue: Journal of structural biology

Bacteria can be propelled in liquids by flagellar filaments that are attached to and moved by flagellar motors. These motors are rotary nanomachines that use the electrochemical potential from ion gradients. The motor can spin in both directions with specific proteins regulating the direction in response to chemotactic stimuli. Here we investigated the structure of flagellar motors of Borrelia spirochetes, the causative agents of Lyme disease in humans. We revealed the structure of the motor complex at 4.6-nm resolution by sub-volume averaging of cryo-electron tomograms and subsequently imposing rotational symmetry. This allowed direct visualisation of individual motor components, the connection between the stator and the peptidoglycan as well as filamentous linkers between the stator and the rod. Two different motor assemblies seem to co-exist at a single bacterial pole. While most motors were completely assembled, a smaller fraction appeared to lack part of the C-ring, which plays a role in protein export and switching the directionality of rotation. Our data suggest a novel mechanism that bacteria may use to control the direction of movement.
Methods for identifying and averaging variable molecular conformations in tomograms of actively contracting insect flight muscle.
Venue: Journal of structural biology

During active muscle contraction, tension is generated through many simultaneous, independent interactions between the molecular motor myosin and the actin filaments. The ensemble of myosin motors displays heterogeneous conformations reflecting different mechanochemical steps of the ATPase pathway. We used electron tomography of actively contracting insect flight muscle fast-frozen, freeze substituted, Araldite embedded, thin-sectioned and stained, to obtain 3D snapshots of the multiplicity of actin-attached myosin structures. We describe procedures for alignment of the repeating lattice of sub-volumes (38.7 nm cross-bridge repeats bounded by troponin) and multivariate data analysis to identify self-similar repeats for computing class averages. Improvements in alignment and classification of repeat sub-volumes reveals (for the first time in active muscle images) the helix of actin subunits in the thin filament and the troponin density with sufficient clarity that a quasiatomic model of the thin filament can be built into the class averages independent of the myosin cross-bridges. We show how quasiatomic model building can identify both strong and weak myosin attachments to actin. We evaluate the accuracy of image classification to enumerate the different types of actin-myosin attachments.
Conformational changes associated with cofactor/substrate binding of 6-phosphogluconate dehydrogenase from Escherichia coli and Klebsiella pneumoniae: Implications for enzyme mechanism.
Venue: Journal of structural biology

6-Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway, catalyzes the oxidative decarboxylation of 6-phosphogluconate, making ribulose 5-phosphate, along with the reduction of NADP(+) to NADPH and the release of CO(2). Here, we report the first apo-form crystal structure of the pathogenic Klebsiella pneumoniae 6PGDH (Kp6PGDH) and the structures of the highly homologous Escherichia coli K12 6PGDH (Ec6PGDH) complexed with substrate, substrate/NADPH and glucose at high resolution. The binding of NADPH to one subunit of the homodimeric structure triggered a 10 degrees rotation and resulting in a 7A movement of the coenzyme-binding domain. The coenzyme was thus trapped in a closed enzyme conformation, in contrast to the open conformation of the neighboring subunit. Comparison of our Ec/Kp6PGDH structures with those of other species illustrated how the domain conformation can be affected upon binding of the coenzyme, which in turn gives rise to concomitant movements of two important NADP(+)-interacting amino acids, M14 and N102. We propose that the catalysis follows an ordered binding mechanism with alternating conformational changes in the corresponding subunits, involving several related amino acid residues.
Radiation stability of proteinase K crystals grown by LB nanotemplate method.
Venue: Journal of structural biology

A detailed analysis of structural and intensity changes induced by X-ray radiation is presented for two types of proteinase K crystals: crystal grown by classical hanging drop method and those grown by Langmuir-Blodgett (LB) nanotemplate. The comparison of various parameters (e.g. intensity per sigma ratio, unit-cell volume, number of unique reflections, B-factors) and electron density maps as a function of radiation dose, demonstrates that crystals, grown by the LB nanotemplate method, appear to be more resistant against radiation damage than crystals grown by the classical hanging drop method.
Monolithic microfluidic mixing-spraying devices for time-resolved cryo-electron microscopy.
Venue: Journal of structural biology

The goal of time-resolved cryo-electron microscopy is to determine structural models for transient functional states of large macromolecular complexes such as ribosomes and viruses. The challenge of time-resolved cryo-electron microscopy is to rapidly mix reactants, and then, following a defined time interval, to rapidly deposit them as a thin film and freeze the sample to the vitreous state. Here we describe a methodology in which reaction components are mixed and allowed to react, and are then sprayed onto an EM grid as it is being plunged into cryogen. All steps are accomplished by a monolithic, microfabricated silicon device that incorporates a mixer, reaction channel, and pneumatic sprayer in a single chip. We have found that microdroplets produced by air atomization spread to sufficiently thin films on a millisecond time scale provided that the carbon supporting film is made suitably hydrophilic. The device incorporates two T-mixers flowing into a single channel of four butterfly-shaped mixing elements that ensure effective mixing, followed by a microfluidic reaction channel whose length can be varied to achieve the desired reaction time. The reaction channel is flanked by two ports connected to compressed humidified nitrogen gas (at 50 psi) to generate the spray. The monolithic mixer-sprayer is incorporated into a computer-controlled plunging apparatus. To test the mixing performance and the suitability of the device for preparation of biological macromolecules for cryo-EM, ribosomes and ferritin were mixed in the device and sprayed onto grids. Three-dimensional reconstructions of the ribosomes demonstrated retention of native structure, and 30S and 50S subunits were shown to be capable of reassociation into ribosomes after passage through the device.
Bio-sintering processes in hexactinellid sponges: fusion of bio-silica in giant basal spicules from Monorhaphis chuni.
Venue: Journal of structural biology

The two sponge classes, Hexactinellida and Demospongiae, comprise a skeleton that is composed of siliceous skeletal elements (spicules). Spicule growth proceeds by appositional layering of lamellae that consist of silica nanoparticles, which are synthesized via the sponge-specific enzyme silicatein. While in demosponges during maturation the lamellae consolidate to a solid rod, the lamellar organization of hexactinellid spicules largely persists. However, the innermost lamellae, near the spicule core, can also fuse to a solid axial cylinder. Similar to the fusion of siliceous nanoparticles and lamella, in several hexactinellid species individual spicules unify during sintering-like processes. Here, we study the different stages of a process that we termed bio-sintering, within the giant basal spicule (GBS) of Monorhaphis chuni. During this study, a major GBS protein component (27 kDa) was isolated and analyzed by MALDI-TOF-MS. The sequences were used to isolate and clone the encoding cDNA via degenerate primer PCR. Bioinformatic analyses revealed a significant sequence homology to silicatein. In addition, the native GBS protein was able to mediate bio-silica synthesis in vitro. We conclude that the syntheses of bio-silica in M. chuni, and the subsequent fusion of nanoparticles to lamellae, and finally to spicules, are enzymatically-driven by a silicatein-like protein. In addition, evidence is now presented that in hexactinellids those fusions involve sintering-like processes.
Contrast transfer function correction applied to cryo-electron tomography and sub-tomogram averaging
Venue: Journal of structural biology

Cryo-electron tomography together with averaging of sub-tomograms containing identical particles can reveal the structure of proteins or protein complexes in their native environment. The resolution of this technique is limited by the contrast transfer function (CTF) of the microscope. The CTF is not routinely corrected in cryo-electron tomography because of difficulties including CTF detection, due to the low signal to noise ratio, and CTF correction, since images are characterised by a spatially variant CTF. Here we simulate the effects of the CTF on the resolution of the final reconstruction, before and after CTF correction, and consider the effect of errors and approximations in defocus determination. We show that errors in defocus determination are well tolerated when correcting a series of tomograms collected at a range of defocus values. We apply methods for determining the CTF parameters in low signal to noise images of tilted specimens, for monitoring defocus changes using observed magnification changes, and for correcting the CTF prior to reconstruction. Using bacteriophage PRD1 as a test sample, we demonstrate that this approach gives an improvement in the structure obtained by sub-tomogram averaging from cryo-electron tomograms.
Supramolecular assembly of collagen fibrils into collagen fiber in fish scales of red seabream, Pagrus major.
Venue: Journal of structural biology

Supramolecular assembly of collagen fibrils into collagen fiber and its distribution in fish scales of red seabream, Pagrus major, were investigated. By virtue of Zernike phase-contrast hard X-ray microscopy, it has been firstly observed that collagen fiber consists of helical substructures of collagen fibrils wrapped with incrustation. As it close to the scalar focus (that is, with aging), loosened- and deteriorated-helical assemblies started to be observed with loosing wrapping incrustation, indicative of the distortion of the basic helical assembly. Various distributions and packing arrangements of collagen fibers were observed dependent on subdivisions of fish scale. Freshly growing edge region of fish scale, embedded into fish skin, showed rarely patched and one directionally arranged collagen fibers, in which specifically triple helical assemblies of collagen fibrils were found. On the contrary, relatively aged region of the rostral field close to the scalar focus displayed randomly directed and densely packed collagen fibers, in which loosened- and deteriorated-helical assemblies of collagen fibrils were mostly found. Our results have demonstrated that hard X-ray microscope can be a powerful tool to study in situ internal structure of biological specimens in an atmospheric pressure.
Collagen matrix remodeling in 3-dimensional cellular space resolved using second harmonic generation and multiphoton excitation fluorescence.
Venue: Journal of structural biology

The structural remodeling of collagen is important in biological processes such as fibrosis, developmental morphogenesis and wound repair. Highly ordered collagen macromolecules produce second harmonic generation signals without the need for any exogenous label. Conversely, the cellular components stained with exogenous labels generate multiphoton excitation fluorescence signals. Both these signals can be captured simultaneously to provide spatially resolved structural reorganization of a collagen matrix and cells. This study dealt with an in vitro collagen gel contraction model of wound repair, in which fibroblasts are seeded into a 3-dimensional type I collagen matrix. When cells are stimulated to trigger collagen contraction, we found the fibroblasts to be highly elongated as well as interconnected in 2-dimensional space, and the collagen, in the form of a visibly clear fibril structure, accumulated around the cells. In the absence of contraction, on the other hand, the cells were predominantly round in shape and no sign of collagen accumulation around the cell was evident despite the presence of the fibrillar collagen morphology in the matrix. Our data suggest second harmonic and multiphoton excitation fluorescence signals can be used in tandem to provide spatially resolved 3-dimensional structural remodeling of a collagen matrix during wound repair.