nils-herrmann/clean_scirepeval huggingface dataset:
Internal structure of sponge glass fiber revealed by ptychographic nanotomography.
Venue: Journal of structural biology

Sponge glass spicules have solicited great interest due to their mechanical and optical properties. Herein we use ptychographic nanotomography to obtain detailed insights into the internal structure of an anchor spicule from the Venus flower basket. The obtained dataset has 90nm resolution in 3D and provides quantitative determination of the electron density. The data reveal significant variations in electron density across the spicule. The central organic filament is found to be slightly but significantly displaced from the spicule central axis. Analysis of the electron density affords an estimate of a protein volume fraction in the organic filament of about 70%. In the highly mineralized part of the spicule, the electron density is seen to display circular symmetry and be neigh independent of position along the spicule long axis. Variations in the electron density beyond those included in current models of spicule mechanics are observed.
Accurate prediction of helix interactions and residue contacts in membrane proteins.
Venue: Journal of structural biology

Accurate prediction of intra-molecular interactions from amino acid sequence is an important pre-requisite for obtaining high-quality protein models. Over the recent years, remarkable progress in this area has been achieved through the application of novel co-variation algorithms, which eliminate transitive evolutionary connections between residues. In this work we present a new contact prediction method for α-helical transmembrane proteins, MemConP, in which evolutionary couplings are combined with a machine learning approach. MemConP achieves a substantially improved accuracy (precision: 56.0%, recall: 17.5%, MCC: 0.288) compared to the use of either machine learning or co-evolution methods alone. The method also achieves 91.4% precision, 42.1% recall and a MCC of 0.490 in predicting helix-helix interactions based on predicted contacts. The approach was trained and rigorously benchmarked by cross-validation and independent testing on up-to-date non-redundant datasets of 90 and 30 experimental three dimensional structures, respectively. MemConP is a standalone tool that can be downloaded together with the associated training data from
Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins
Venue: Journal of structural biology

We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization.
Structural characterization of GASDALIE Fc bound to the activating Fc receptor FcγRIIIa.
Venue: Journal of structural biology

The Fc region of Immunoglobulin G (IgG) initiates inflammatory responses such as antibody-dependent cell-mediated cytotoxicity (ADCC) through binding to activating Fc receptors (FcγRI, FcγRIIa, FcγRIIIa). These receptors are expressed on the surface of immune cells including macrophages, dendritic cells, and natural killer cells. An inhibitory receptor, FcγRIIb, is expressed on macrophages and other myeloid leukocytes simultaneously with the activating receptor FcγRIIa, thereby setting a threshold for cell activation. The affinity of IgG Fc for binding activating Fc receptors depends on IgG subclass and the composition of N-linked glycans attached to a conserved asparagine in the Fc CH2 domain. For example, Fc regions with afucosylated glycans bind more tightly to FcγRIIIa than fucosylated Fc, and afucosylated Fcs exhibit enhanced ADCC activity in vivo and in vitro. Enhanced pro-inflammatory responses have also been seen for Fc regions with amino acid substitutions. GASDALIE Fc is an Fc mutant (G236A/S239D/A330L/I332E) that exhibits a higher affinity for FcγRIIIa and increased effector functions in vivo compared to wild-type Fc. To explore its altered functions, we compared the affinities of GASDALIE and wild-type Fc for activating and inhibitory FcγRs. We also determined the crystal structure of GASDALIE Fc alone and bound to FcγRIIIa. The overall structure of GASDALIE Fc alone was similar to wild-type Fc structures, however, increased electrostatic interactions in the GASDALIE Fc:FcγRIIIa interface compared with other Fc:FcγR structures suggest a mechanism for the increased affinity of GASDALIE Fc for FcγRIIIa.
Helicobacter pylori cell binding factor 2: Insights into domain motion.
Venue: Journal of structural biology

Helicobacter pylori cell binding factor 2 (HpCBF2) is an antigenic virulence factor belonging to the SurA-like peptidyl-prolyl cis-trans isomerase family with implications for pathogenicity in the human gastrointestinal tract. HpCBF2 possesses PPIase activity and could act as a periplasmic chaperone to regulate outer membrane protein assembly. Here, we measured the isomerization and chaperone activity of HpCBF2, and determined the crystal structure of HpCBF2 in complex with an inhibitor, indole-2-carboxylic acid (I2CA), at 2.4Å resolution. HpCBF2-I2CA forms a homodimer encasing a large central hydrophobic cavity with a basket-like structure, and each monomer contains a PPIase and a chaperone domain. In the HpCBF2-I2CA dimer, the two PPIase domains separate by a distance of 22.8Å, while the two chaperone domains arrange in a domain-swap manner. The PPIase domains bound with I2CA ligand face towards the chaperone domains and are shielded by surrounding hydrophobic residues. With the aid of SAXS experiments, we also revealed domain motion between the apo- and I2CA-bound states of HpCBF2. The domain motion in HpCBF2 might be necessary for the isomerization activity of PPIase and the accommodation of the unfolded and partially folded peptides to refold by chaperone domain.
Structure of wild type and mutant F508del CFTR: A small-angle X-ray scattering study of the protein-detergent complexes.
Venue: Journal of structural biology

CFTR is an anionic channel expressed in epithelia whose mutations cause cystic fibrosis. Wild (WT) and mutated (F508del) types were over-expressed in yeast, solubilised in the detergent LPG-14 and purified. The detergent-CFTR complexes were studied by SAXS techniques using a solvent of variable density. The final result of the study is the numerical value of a set of parameters: molecular mass, volume and radius of gyration, average electron density and second moment of the electron density fluctuations inside the particles. It is also shown that in the complex the centres of gravity of CFTR and of the detergent are displaced relative to each other. The analysis of these parameters led to the determination of the size and shape of the volumes occupied by protein and by detergent in the complex. WT-CFTR to be an elongated molecule (maximum diameter ∼12.4nm) which spans a flat detergent micelle. The distance distribution function, P(r) confirms that the WT-CFTR is elongated and with an inhomogeneous electronic density. The F508del-CFTR molecule is also elongated (maximum diameter ∼13.2nm), but the associated detergent micelle hides a larger surface, plausibly related to an increased exposure of hydrophobic portions of the mutated protein. The corresponding P(r) is consistent with the presence of well defined domains, probably linked by flexible regions. These differences suggest that the full-length mutant F508del-CFTR has a detectably different conformation, in contrast to the minor differences observed for the isolated F508-containing domain. We interpret the data in terms of an incomplete post-translational assembly of the protein domains.
Statistical modeling and removal of lipid membrane projections for cryo-EM structure determination of reconstituted membrane proteins.
Venue: Journal of structural biology

This paper describes steps in the single-particle cryo-EM 3D structure determination of membrane proteins in their membrane environment. Using images of the Kv1.2 potassium-channel complex reconstituted into lipid vesicles, we describe procedures for the merging of focal-pairs of exposures and the removal of the vesicle-membrane signal from the micrographs. These steps allow 3D reconstruction to be performed from the protein particle images. We construct a 2D statistical model of the vesicle structure based on higher-order singular value decomposition (HOSVD), by taking into account the structural symmetries of the vesicles in polar coordinates. Non-roundness in the vesicle structure is handled with a non-linear shape alignment to a reference, which ensures a compact model representation. The results show that the learned model is an accurate representation of the imaged vesicle structures. Precise removal of the strong membrane signals allows better alignment and classification of images of small membrane-protein particles, and allows higher-resolution 3D reconstruction.
Biomineralization pathways in a foraminifer revealed using a novel correlative cryo-fluorescence-SEM-EDS technique.
Venue: Journal of structural biology

Foraminifera are marine protozoans that are widespread in oceans throughout the world. Understanding biomineralization pathways in foraminifera is particularly important because their calcitic shells are major components of global calcium carbonate production. We introduce here a novel correlative approach combining cryo-SEM, cryo-fluorescence imaging and cryo-EDS. This approach is applied to the study of ion transport processes in the benthic foraminifer genus Amphistegina. We confirm the presence of large sea water vacuoles previously identified in intact and partially decalcified Amphistegina lobifera specimens. We observed relatively small vesicles that were labelled strongly with calcein, and also identified magnesium (Mg)-rich mineral particles in the cytoplasm, as well as in the large sea water vacuoles. The combination of cryo-microscopy with elemental microanalysis and fluorescence imaging reveals new aspects of the biomineralization pathway in foraminifera which are, to date, unique in the world of biomineralization. This approach is equally applicable to the study of biomineralization pathways in other organisms.
Supramolecular structure of enterobacterial wild-type lipopolysaccharides (LPS), fractions thereof, and their neutralization by Pep19-2.5.
Venue: Journal of structural biology

Lipopolysaccharides (LPS) belong to the strongest immune-modulating compounds known in nature, and are often described as pathogen-associated molecular patterns (PAMPs). In particular, at higher concentrations they are responsible for sepsis and the septic shock syndrome associated with high lethality. Since most data are indicative that LPS aggregates are the bioactive units, their supramolecular structures are considered to be of outmost relevance for deciphering the molecular mechanisms of its bioactivity. So far, however, most of the data available addressing this issue, were published only for the lipid part (lipid A) and the core-oligosaccharide containing rough LPS, representing the bioactive unit. By contrast, it is well known that most of the LPS specimen identified in natural habitats contain the smooth-form (S-form) LPS, which carry additionally a high-molecular polysaccharide (O-chain). To fill this lacuna and going into a more natural system, here various wild-type (smooth form) LPS including also some LPS fractions were investigated by small-angle X-ray scattering with synchrotron radiation to analyze their aggregate structure. Furthermore, the influence of a recently designed synthetic anti-LPS peptide (SALP) Pep19-2.5 on the aggregate structure, on the binding thermodynamics, and on the cytokine-inducing activity of LPS were characterized, showing defined aggregate changes, high affinity binding and inhibition of cytokine secretion. The data obtained are suitable to refine our view on the preferences of LPS for non-lamellar structures, representing the highest bioactive forms which can be significantly influenced by the binding with neutralizing peptides such as Pep19-2.5.
A new structural model of Alzheimer's Aβ42 fibrils based on electron paramagnetic resonance data and Rosetta modeling.
Venue: Journal of structural biology

Brain deposition of Aβ in the form of amyloid plaques is a pathological hallmark of Alzheimer's disease. There are two major species of Aβ in the brain: Aβ42 and Aβ40. Although Aβ40 is several-fold more abundant than Aβ42 in soluble form, Aβ42 is the major component of amyloid plaques. Structural knowledge of Aβ42 fibrils is important both for understanding the process of Aβ aggregation and for designing fibril-targeting drugs. Here we report site-specific structural information of Aβ42 fibrils at 22 residue positions based on electron paramagnetic resonance data. In combination with structure prediction program Rosetta, we modeled Aβ42 fibril structure at atomic resolution. Our Aβ42 fibril model consists of four parallel in-register β-sheets: βN (residues ∼7-13), β1 (residues ∼17-20), β2 (residues ∼32-36), and βC (residues 39-41). The region of β1-loop-β2 in Aβ42 fibrils adopts similar structure as that in Aβ40 fibrils. This is consistent with our cross seeding data that Aβ42 fibril seeds shortened the lag phase of Aβ40 fibrillization. On the other hand, Aβ42 fibrils contain a C-terminal β-arc-β motif with a special turn, termed "arc", at residues 37-38, which is absent in Aβ40 fibrils. Our results can explain both the higher aggregation propensity of Aβ42 and the importance of Aβ42 to Aβ40 ratio in the pathogenesis of Alzheimer's disease.
A detailed look at the cytoskeletal architecture of the Giardia lamblia ventral disc.
Venue: Journal of structural biology

Giardia lamblia is a protistan parasite that infects and colonizes the small intestine of mammals. It is widespread and particularly endemic in the developing world. Here we present a detailed structural study by 3-D negative staining and cryo-electron tomography of a unique Giardia organelle, the ventral disc. The disc is composed of a regular array of microtubules and associated sheets, called microribbons that form a large spiral, held together by a myriad of mostly unknown associated proteins. In a previous study we analyzed by cryo-electron tomography the central microtubule portion (here called disc body) of the ventral disc and found a large portion of microtubule associated inner (MIPs) and outer proteins (MAPs) that render these microtubules hyper-stable. With this follow-up study we expanded our 3-D analysis to different parts of the disc such as the ventral and dorsal areas of the overlap zone, as well as the outer disc margin. There are intrinsic location-specific characteristics in the composition of microtubule-associated proteins between these regions, as well as large differences between the overall architecture of microtubules and microribbons. The lateral packing of microtubule-microribbon complexes varies substantially, and closer packing often comes with contracted lateral tethers that seem to hold the disc together. It appears that the marginal microtubule-microribbon complexes function as outer, laterally contractible lids that may help the cell to clamp onto the intestinal microvilli. Furthermore, we analyzed length, quantity, curvature and distribution between different zones of the disc, which we found to differ from previous publications.
Spiculogenesis in the siliceous sponge Lubomirskia baicalensis studied with fluorescent staining.
Venue: Journal of structural biology

Siliceous sponges are the most primitive multicellular animals whose skeleton consists of spicules - needle-like constructions from silicon dioxide surrounding organic axial filaments. Mechanisms of spicule formation have been intensively studied due to the high ecological importance of sponges and their interest to materials science. Light and electron microscopy are not appropriate enough to display the process from silicon-enriched cells to mature spicules because of composite structure of the sponge tissues. In this article, spiculogenesis in the siliceous sponge has been studied for the first time with the use of fluorescent microscopy. Fluorescent vital dye NBD-N2 was applied to stain growing siliceous structures in the sponge and primmorph cell system. The main stages of spicule growth in the fresh-water sponge Lubomirskia baicalensis (Pallas, 1773) were visualized: silicon accumulation in sclerocytes; formation of an organic filament protruding from the cell; further elongation of the filament and growth of the spicule in a spindle-like form with enlargement in the center; merger with new sclerocytes and formation of the mature spicule. Fluorescent microscopy combined with SEM allows us to overcome the virtual differentiation between intra- and extracellular mechanisms of spicule growth. The growing spicule can capture silicic acid from the extracellular space and merge with new silicon-enriched cells. Visualization of the growing spicules with the fluorescent dye allows us to monitor sponge viability in ecological or toxicological experiments and to apply genomic, proteomic and biochemical techniques.
Tectorins crosslink type II collagen fibrils and connect the tectorial membrane to the spiral limbus.
Venue: Journal of structural biology

All inner ear organs possess extracellular matrix appendices over the sensory epithelia that are crucial for their proper function. The tectorial membrane (TM) is a gelatinous acellular membrane located above the hearing sensory epithelium and is composed mostly of type II collagen, and α and β tectorins. TM molecules self-assemble in the endolymph fluid environment, interacting medially with the spiral limbus and distally with the outer hair cell stereocilia. Here, we used immunogold labeling in freeze-substituted mouse cochleae to assess the fine localization of both tectorins in distinct TM regions. We observed that the TM adheres to the spiral limbus through a dense thin matrix enriched in α- and β-tectorin, both likely bound to the membranes of interdental cells. Freeze-etching images revealed that type II collagen fibrils were crosslinked by short thin filaments (4±1.5nm, width), resembling another collagen type protein, or chains of globular elements (15±3.2nm, diameter). Gold-particles for both tectorins also localized adjacent to the type II collagen fibrils, suggesting that these globules might be composed essentially of α- and β-tectorins. Finally, the presence of gold-particles at the TM lower side suggests that the outer hair cell stereocilia membrane has a molecular partner to tectorins, probably stereocilin, allowing the physical connection between the TM and the organ of Corti.
Optimizing force spectroscopy by modifying commercial cantilevers: Improved stability, precision, and temporal resolution.
Venue: Journal of structural biology

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) enables a wide array of studies, from measuring the strength of a ligand-receptor bond to elucidating the complex folding pathway of individual membrane proteins. Such SMFS studies and, more generally, the diverse applications of AFM across biophysics and nanotechnology are improved by enhancing data quality via improved force stability, force precision, and temporal resolution. For an advanced, small-format commercial AFM, we illustrate how these three metrics are limited by the cantilever itself rather than the larger microscope structure, and then describe three increasingly sophisticated cantilever modifications that yield enhanced data quality. First, sub-pN force precision and stability over a broad bandwidth (Δf=0.01-20Hz) is routinely achieved by removing a long (L=100μm) cantilever's gold coating. Next, this sub-pN bandwidth is extended by a factor of ∼50 to span five decades of bandwidth (Δf=0.01-1000Hz) by using a focused ion beam (FIB) to modify a shorter (L=40μm) cantilever. Finally, FIB-modifying an ultrashort (L=9μm) cantilever improves its force stability and precision while maintaining 1-μs temporal resolution. These modified ultrashort cantilevers have a reduced quality factor (Q≈0.5) and therefore do not apply a substantial (30-90pN), high-frequency force modulation to the molecule, a phenomenon that is unaccounted for in traditional SMFS analysis. Currently, there is no perfect cantilever for all applications. Optimizing AFM-based SMFS requires understanding the tradeoffs inherent to using a specific cantilever and choosing the one best suited to a particular application.
Loop-to-helix transition in the structure of multidrug regulator AcrR at the entrance of the drug-binding cavity.
Venue: Journal of structural biology

Multidrug transcription regulator AcrR from Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 belongs to the tetracycline repressor family, one of the largest groups of bacterial transcription factors. The crystal structure of dimeric AcrR was determined and refined to 1.56Å resolution. The tertiary and quaternary structures of AcrR are similar to those of its homologs. The multidrug binding site was identified based on structural alignment with homologous proteins and has a di(hydroxyethyl)ether molecule bound. Residues from helices α4 and α7 shape the entry into this binding site. The structure of AcrR reveals that the extended helical conformation of helix α4 is stabilized by the hydrogen bond between Glu67 (helix α4) and Gln130 (helix α7). Based on the structural comparison with the closest homolog structure, the Escherichia coli AcrR, we propose that this hydrogen bond is responsible for control of the loop-to-helix transition within helix α4. This local conformational switch of helix α4 may be a key step in accessing the multidrug binding site and securing ligands at the binding site. Solution small-molecule binding studies suggest that AcrR binds ligands with their core chemical structure resembling the tetracyclic ring of cholesterol.
A new crystal form of human transthyretin obtained with a curcumin derived ligand.
Venue: Journal of structural biology

Transthyretin (TTR), a 54kDa homotetrameric protein that transports thyroxine (T4), has been associated with clinical cases of TTR amyloidosis for its tendency to aggregate to form fibrils. Many ligands with a potential to inhibit fibril formation have been studied by X-ray crystallography in complex with TTR. Unfortunately, the ligand is often found in ambiguous electron density that is difficult to interpret. The ligand validation statistics suggest over-interpretation, even for the most active compounds like diflunisal. The primary technical reason is its position on a crystallographic 2-fold axis in the most common crystal form. Further investigations with the use of polyethylene glycol (PEG) to crystallize TTR complexes have resulted in a new trigonal polymorph with two tetramers in the asymmetric unit. The ligand used to obtain this new polymorph, 4-hydroxychalcone, is related to curcumin. Here we evaluate this crystal form to understand the contribution it may bring to the study of TTR ligands complexes, which are often asymmetric.