nils-herrmann/clean_scirepeval huggingface dataset:
Crystallographic characterization of the crossed lamellar structure in the bivalve Meretrix lamarckii using electron beam techniques.
Venue: Journal of structural biology

To understand the formation mechanism of crossed lamellar structures in molluskan shells, the crystallographic structural features in the shell of a bivalve, Meretrix lamarckii, were investigated using scanning electron microscopy, electron backscattered diffraction, and transmission electron microscopy with a focused ion beam sample preparation technique. Approximately 0.5 μm-thick lamellae (the second-order units) are piled up obliquely toward the growth direction to form the first-order unit and the obliquity is inverted between adjacent units along the shell thickness direction. The first-order units originate around the center of the shell, initially growing parallel to the shell and subsequently curving toward the inner or outer surfaces. The lamellae consist of aragonite granular and columnar layers, which group together to adopt the same crystal orientation forming crystallographic units (crystallites). Multiple {110} twins are common both in the granular and columnar layers. The crystallite c-axis is parallel to the columns and is inclined at angles 0-50° from the lamellar normal (dispersing among individual lamellae), toward the shell growth direction. Probably, the directions of the a- and b-axes are random in the lamellae, showing no specific orientation.
Edged watershed segmentation: a semi-interactive algorithm for segmentation of low-resolution maps from electron cryomicroscopy.
Venue: Journal of structural biology

Electron cryomicroscopy (cryo-EM) allows for the structural analysis of large protein complexes that may be difficult to study by other means. Frequently, maps of complexes from cryo-EM are obtained at resolutions between 10 and 25Å. To aid in the interpretation of these medium- to low-resolution maps, they may be subdivided into three-dimensional segments representing subunits or subcomplexes. This division is often accomplished using a manual segmentation approach. While extremely useful, manual segmentation is subjective. We have developed a novel semi-interactive segmentation algorithm that can incorporate prior knowledge of subunit composition or structure without biasing the boundaries between subunits or subcomplexes. This algorithm has been characterized with experimental and simulated cryo-EM density maps at resolutions between 10 and 25Å.
An adaptation of the Wiener filter suitable for analyzing images of isolated single particles.
Venue: Journal of structural biology

The Wiener filter is a standard means of optimizing the signal in sums of aligned, noisy images obtained by electron cryo-microscopy (cryo-EM). However, estimation of the resolution-dependent ("spectral") signal-to-noise ratio (SSNR) from the input data has remained problematic, and error reduction due to specific application of the SSNR term within a Wiener filter has not been reported. Here we describe an adjustment to the Wiener filter for optimal summation of images of isolated particles surrounded by large regions of featureless background, as is typically the case in single-particle cryo-EM applications. We show that the density within the particle area can be optimized, in the least-squares sense, by scaling the SSNR term found in the conventional Wiener filter by a factor that reflects the fraction of the image field occupied by the particle. We also give related expressions that allow the SSNR to be computed for application in this new filter, by incorporating a masking step into a Fourier Ring Correlation (FRC), a standard resolution measure. Furthermore, we show that this masked FRC estimation scheme substantially improves on the accuracy of conventional SSNR estimation methods. We demonstrate the validity of our new approach in numeric tests with simulated data corresponding to realistic cryo-EM imaging conditions. This variation of the Wiener filter and accompanying derivation should prove useful for a variety of single-particle cryo-EM applications, including 3D reconstruction.
Solution structure of HtrA PDZ domain from Streptococcus pneumoniae and its interaction with YYF-COOH containing peptides.
Venue: Journal of structural biology

High-temperature requirement A (HtrA), a highly conserved family of serine protease, plays crucial roles in protein quality control in prokaryotes and eukaryotes. The HtrA protein contains a C-terminal PDZ domain that mediates the proteolytic activity. Here we reported the solution structure of the HtrA PDZ domain from Streptococcus pneumoniae by NMR spectroscopy. Our results showed that the structure of HtrA PDZ domain, which contains three α-helices and five β-strands, illustrates conservation within the canonical PDZ domains. In addition, we demonstrated the interactions between S. pneumoniae HtrA PDZ domain and peptides with the motif XXX-YYF-COOH by surface plasmon resonance. Besides, we identified the ligand binding surface and the critical residues responsible for ligand binding of HtrA PDZ domain by chemical shift perturbation and site-directed mutagenesis.
The unusual extended C-terminal helix of the peroxisomal α/β-hydrolase Lpx1 is involved in dimer contacts but dispensable for dimerization.
Venue: Journal of structural biology

The yeast peroxisomal hydrolase Lpx1 belongs to the α/β-hydrolase superfamily. In the absence of Lpx1, yeast peroxisomes show an aberrant vacuolated morphology similar to what is found in peroxisomal disorder patients. Here, we present the crystal structure of Lpx1 determined at a resolution of 1.9 Å. The structure reveals the complete catalytic triad with an unusual location of the acid residue after strand β6 of the canonical α/β-hydrolase fold. A four-helix cap domain covers the active site. The interface between the α/β-hydrolase core and the cap domain forms the potential substrate binding site, which may also comprise the tunnel that leads into the protein interior and widens into a cavity. Two further tunnels connect the active site to the protein surface, potentially facilitating substrate access. Lpx1 is a homodimer. The α/β-hydrolase core folds of the two protomers form the dimer contact site. Further dimerization contacts arise from the mutual embracement of the cap domain of one protomer by the non-canonical C-terminal helix of the other, resulting in a total buried surface area of some 6000 Å². The unusual C-terminal helix sticks out from the core fold to which it is connected by an extended flexible loop. We analyzed whether this helix is required for dimerization and for import of the dimer into peroxisomes using biochemical assays in vitro and a microscopy-based interaction assay in mammalian cells. Surprisingly, the C-terminal helix is dispensable for dimerization and dimer import. The unusually robust self-interaction suggests that Lpx1 is imported into peroxisomes as dimer.
Stability of gp41 hairpin and helix bundle assembly probed by combined stacking and circular dichroic approaches.
Venue: Journal of structural biology

The envelope glycoprotein gp41 of HIV-1 undergoes structural rearrangement to form a helix hairpin during the virus-mediated fusion. Previous studies to investigate the folding and stability of hairpin did not monitor the end-to-end distance of the molecule. To directly probe the distance change, rhodamine dye was conjugated to the gp41 recombinant near the N- and C-terminal regions to detect the UV absorption and fluorescence intensity changes induced by the chemical denaturant guanidinium chloride (GdmCl). Using the singly- and doubly-labeled constructs allowed us to distinguish between the hairpin formation and protein oligomerization. A biphasic transition of helical structure for the wild type protein was revealed by circular dichroism measurements while unfolding of the hairpin occurred at 6M GdmCl. The relevance of our study to the fusion inhibitor for HIV-1 was borne out by results on the mutants at the positions within the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) regions. A monophasic transition at lower denaturant concentration was detected for the NHR mutant supporting the concept of differential stability of NHR and CHR helical structure. The conclusion that the observed unstacking of doubly-labeled variant arises principally from the intra-molecular dimers was drawn from the unstacking of the protein labeled in the loop. Remarkably, it is deduced that the hairpin is more stable than the CHR helical structure. A model for denaturation of the helix hairpin bundle was proposed from these results. The biological implications of the findings and further applications of the distance-based approach were discussed.
The canine baculum: the structure and mechanical properties of an unusual bone.
Venue: Journal of structural biology

The baculum is an extraskeletal bone located in the penis of a few species in several orders of mammals such as carnivores, insectivores, rodents, bats and primates. This study aims to describe the structure, architecture and mechanical properties of the canine baculum. To this end canine bacula from castrated and uncastrated dogs were collected and examined by light microscopy, micro-computed tomography (microCT) scanning, histological staining, and mechanical testing. Their mineral density and mechanical properties were compared with those of a typical skeletal bone (the radius) in the same dog. Furthermore, a numerical model of a representative baculum was created and its mechanical performance analyzed using the finite element method, in order to try to elucidate its function. Examination of light microscopy images of transverse sections shows that the baculum consists of a typical sandwich structure, with two cortical plates separated, and joined, by loose cancellous bone. MicroCT scans reveal that the mineral density is lower in the baculum than in the radius, both in castrated as well as in uncastrated dogs, resulting in much lower stiffness. Castration was found to decrease the mineral density in both the baculum and the radius. The most likely function of the baculum of the dog is to stiffen the penis to assist intromission, and its much lower mineral density compared to that of the radius may be a mechanism designed to decrease the stiffness somewhat, and thus reduce the risk of fracture during copulation.
Reference-free particle selection enhanced with semi-supervised machine learning for cryo-electron microscopy.
Venue: Journal of structural biology

Reference-based methods have dominated the approaches to the particle selection problem, proving fast, and accurate on even the most challenging micrographs. A reference volume, however, is not always available and compiling a set of reference projections from the micrographs themselves requires significant effort to attain the same level of accuracy. We propose a reference-free method to quickly extract particles from the micrograph. The method is augmented with a new semi-supervised machine-learning algorithm to accurately discriminate particles from contaminants and noise.
Computer controlled cryo-electron microscopy--TOM² a software package for high-throughput applications.
Venue: Journal of structural biology

Automated data acquisition expedites structural studies by electron microscopy and it allows to collect data sets of unprecedented size and consistent quality. In electron tomography it greatly facilitates the systematic exploration of large cellular landscapes and in single particle analysis it allows to generate data sets for an exhaustive classification of coexisting molecular states. Here we describe a novel software philosophy and architecture that can be used for a great variety of automated data acquisition scenarios. Based on our original software package TOM, the new TOM(2) package has been designed in an object-oriented way. The whole program can be seen as a collection of self-sufficient modules with defined relationships acting in a concerted manner. It subdivides data acquisition into a set of hierarchical tasks, bonding data structure and the operations to be performed tightly together. To demonstrate its capacity for high-throughput data acquisition it has been used in conjunction with instrumentation combining the latest technological achievements in electron optics, cryogenics and robotics. Its performance is demonstrated with a single particle analysis case study and with a batch tomography application.
Tubular frameworks guiding orderly bone formation in the antler of the red deer (Cervus elaphus).
Venue: Journal of structural biology

Deer antler is a bony tissue which re-grows every year after shedding. Growth speed and material properties of this tissue are truly remarkable, making it an interesting model for bone regeneration. Surprisingly, not much is known about the ultrastructure of the calcified tissues and the temporal sequence of their development during antler growth. We use a combination of imaging tools based on light and electron microscopy to characterize antler tissue at various stages of development. We observe that mineralized cartilage is first transformed into a bone framework with low degree of collagen fibril ordering at the micron level. This framework has a honeycomb-like appearance with the cylindrical pores oriented along the main antler axis. Later, this tissue is filled with primary osteons, whose collagen fibrils are mainly oriented along the pores, thus improving the antler's mechanical properties. This strongly suggests that to achieve very fast organ growth it is advantageous to have a longitudinal porous framework as an intermediate step in bone formation. The example of antler shows that geometric features of this framework are crucial, and a tubular geometry with a diameter in the order of hundred micrometers seems to be a good solution for fast framework-mediated bone formation.
Three-dimensional reconstruction using an adaptive simultaneous algebraic reconstruction technique in electron tomography.
Venue: Journal of structural biology

Three-dimensional (3D) reconstruction of electron tomography (ET) has emerged as an important technique in analyzing structures of complex biological samples. However most of existing reconstruction methods are not suitable for extremely noisy and incomplete data conditions. We present an adaptive simultaneous algebraic reconstruction technique (ASART) in which a modified multilevel access scheme and an adaptive relaxation parameter adjustment method are developed to improve the quality of the reconstructed 3D structure. The reconstruction process is facilitated by using a column-sum substitution approach. This modified multilevel access scheme is adopted to arrange the order of projections so as to minimize the correlations between consecutive views within a limited angle range. In the adaptive relaxation parameter adjustment method, not only the weight matrix (as in the existing methods) but the gray levels of the pixels are employed to adjust the relaxation parameters so that the quality of the reconstruction is improved while the convergence process of the reconstruction is accelerated. In the column-sum substitution approach, the computation to obtain the reciprocal of the sum for the columns in each view is avoided so that the needed computations for each iteration can be reduced. Experimental results show that the proposed technique ASART is better based on objective quality measures than other methods, especially when data is noisy and limited in tilt angles. At the same time, the reconstruction by ASART outperforms that of simultaneous algebraic reconstruction technique (SART) in speed.
Crystallographic analysis reveals a novel second binding site for trimethoprim in active site double mutants of human dihydrofolate reductase.
Venue: Journal of structural biology

In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h) DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (K(i) 49 nM) compared to hDHFR (K(i) 4093 nM). Two mutants that contain one substitution from pj--and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show K(i) values of 593 and 617 nM, respectively; these K(i) values are well above both the K(i) for pjDHFR and are similar to pcDHFR (Q35K/N64F and Q35S/N64F) (305nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes.
Molecular recognition force spectroscopy of a specific lectin-carbohydrate interaction at single-molecule level.
Venue: Journal of structural biology

Carbohydrates are involved in many essential biological recognition processes in physiological and pathological states. Thus, it is important to understand the mechanism of protein-carbohydrate interactions at molecular level. In the present study, molecular recognition force spectroscopy was applied to investigate the interactions between RCA₁₂₀, a lectin from Ricinus communis, and galactose (Gal) and asialofetuin (ASF) at the single-molecule level. RCA₁₂₀ coupled to the AFM tip could specifically recognize Gal and ASF, respectively. The unbinding forces of RCA₁₂₀-Gal and RCA₁₂₀-ASF increase linearly with the logarithm of loading rate. The results reveal that the binding capability of RCA₁₂₀ toward Gal is weaker than that of ASF, implicating a multivalent effect in the RCA₁₂₀-ASF interaction.
Experimental evaluation of support vector machine-based and correlation-based approaches to automatic particle selection.
Venue: Journal of structural biology

The goal of this study is to evaluate the performance of software for automated particle-boxing, and in particular the performance of a new tool (TextonSVM) that recognizes the characteristic texture of particles of interest. As part of a high-throughput protocol, we use human editing that is based solely on class-average images to create final data sets that are enriched in what the investigator considers to be true-positive particles. The Fourier shell correlation (FSC) function is then used to characterize the homogeneity of different single-particle data sets that are derived from the same micrographs by two or more alternative methods. We find that the homogeneity is generally quite similar for class-edited data sets obtained by the texture-based method and by SIGNATURE, a cross-correlation-based method. The precision-recall characteristics of the texture-based method are, on the other hand, significantly better than those of the cross-correlation based method; that is to say, the texture-based approach produces a smaller fraction of false positives in the initial set of candidate particles. The computational efficiency of the two approaches is generally within a factor of two of one another. In situations when it is helpful to use a larger number of templates (exemplars), however, TextonSVM scales in a much more efficient way than do boxing programs that are based on localized cross-correlation.
Structure and stability of the lamin A tail domain and HGPS mutant.
Venue: Journal of structural biology

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging syndrome caused by the expression and accumulation of a mutant form of lamin A, Δ50 lamin A. As a component of the cell's nucleoskeleton, lamin A plays an important role in the mechanical stabilization of the nuclear envelope and in other nuclear functions. It is largely unknown how the characteristic 50 amino acid deletion affects the conformation of the mostly intrinsically disordered tail domain of lamin A. Here we perform replica exchange molecular dynamics simulations of the tail domain and determine an ensemble of semi-stable structures. Based on these structures we show that the ZMPSTE 24 cleavage site on the precursor form of the lamin A tail domain orients itself in such a way as to facilitate cleavage during the maturation process. We confirm our simulated structures by comparing the thermodynamic properties of the ensemble structures to in vitro stability measurements. Using this combination of experimental and computational techniques, we compare the size, heterogeneity of size, thermodynamic stability of the Ig-fold, as well as the mechanisms of force-induced denaturation. Our data shows that the Δ50 lamin A tail domain is more compact and displays less heterogeneity than the mature lamin A tail domain. Altogether these results suggest that the altered structure and stability of the tail domain can explain changed protein-protein and protein-DNA interactions and may represent an etiology of the disease. Also, this study provides the first molecular structure(s) of the lamin A tail domain, which is confirmed by thermodynamic tests in experiment.
Mussel collagen molecules with silk-like domains as load-bearing elements in distal byssal threads.
Venue: Journal of structural biology

Mechanically stressed biological materials like tendon, spider silk or mussel byssal threads are typically composite materials comprising multi-domain proteins, in which molecular building blocks contribute to overall material function. Mussel byssal threads are the anchorage of sessile mytilid mussels, which withstand recurring external loads from waves and tides. A single thread is elastic and ductile proximally, while the distal portion exhibits an extraordinary stiffness and toughness with a transient gradient of both mechanical features along the thread. The main components of byssal threads include a set of various collagen-like structural proteins (preCols) consisting of a collagenous core sequence flanked by globular domains. Here, structural analysis using polarized Fourier-transform infrared spectroscopy (FTIR) on stretched distal portions of mussel byssal threads determines the impact of external linear load on various molecular moieties. It is concluded that the preCol collagenous core domain is the main load-bearing element in distal byssal threads, while polyalanine beta-sheets in the flanking domains, similar to those found in spider silk proteins, provide high stiffness at low strains. Load dissipation is mediated by domain stretching of amorphous glycine-rich helical moieties followed by complete unfolding of the preCol flanking domains.