nils-herrmann/clean_scirepeval huggingface dataset:
Crystal structure of the globular domain of C1QTNF5: Implications for late-onset retinal macular degeneration.
Venue: Journal of structural biology

Autosomal dominant late-onset retinal macular degeneration (L-ORMD) is caused by a single S163R mutation in the C1q and tumor necrosis factor-related protein 5 (C1QTNF5) gene. The C1QTNF5 gene encodes a secreted and membrane-associated protein involved in adhesion of retinal pigmented epithelial cells (RPE) to Bruch's membrane. The crystal structure of the trimeric globular domain of human C1QTNF5 at 1.34Å resolution reveals unique features of this novel C1q family member. It lacks a Ca²⁺-binding site, displays a remarkable non-uniform distribution of surface electrostatic potentials and possesses a unique sequence (F₁₈₁F₁₈₂G₁₈₃G₁₈₄W₁₈₅P₁₈₆) that forms a hydrophobic plateau surrounded by Lys and Arg residues with a solvent cavity underneath. S₁₆₃ forms a hydrogen bond with F₁₈₂ in a hydrophobic area extending to the hydrophobic plateau. The pathogenic mutation S163R disrupts this hydrogen bonding and positively charges these hydrophobic areas. Thus, our analysis provides insights into the structural basis of the L-ORMD disease mechanism.
A graph theory method for determination of cryo-EM image focuses.
Venue: Journal of structural biology

Accurate determination of micrograph focuses is essential for averaging multiple images to reach high-resolution 3-D reconstructions in electron cryo-microscopy (cryo-EM). Current methods use iterative fitting of focus-dependent simulated power spectra to the power spectra of experimental images, with the fitting performed independently for different images. Here we have developed a novel graph theory based method in which the rotational average focus and individual angular sector focuses of all images are determined simultaneously in closed form using the least square solution of overdetermined linear equations. The new method was shown to be fast, accurate, and robust in tests with large datasets of experimental low dose cryo-EM images. Its integration with three classic power spectra fitting methods also allows cross validation of the results by these vastly different methods. The new integrated focus determination method will improve reliability of automated focus determination for large-scale data processing that is increasingly common in the cryo-EM field.
Structural characterization of CHCHD5 and CHCHD7: two atypical human twin CX9C proteins.
Venue: Journal of structural biology

Twin CX(9)C proteins constitute a large protein family among all eukaryotes; are putative substrates of the mitochondrial Mia40-dependent import machinery; contain a coiled coil-helix-coiled coil-helix (CHCH) fold stabilized by two disulfide bonds as exemplified by three structures available for this family. However, they considerably differ at the primary sequence level and this prevents an accurate prediction of their structural models. With the aim of expanding structural information on CHCH proteins, here we structurally characterized human CHCHD5 and CHCHD7. While CHCHD5 has two weakly interacting CHCH domains which sample a range of limited conformations as a consequence of hydrophobic interactions, CHCHD7 has a third helix hydrophobically interacting with an extension of helix α2, which is part of the CHCH domain. Upon reduction of the disulfide bonds both proteins become unstructured exposing hydrophobic patches, with the result of protein aggregation/precipitation. These results suggest a model where the molecular interactions guiding the protein recognition between Mia40 and the disulfide-reduced CHCHD5 and CHCHD7 substrates occurs in vivo when the latter proteins are partially embedded in the protein import pore of the outer membrane of mitochondria.
Automatic magnification determination of electron cryomicroscopy images using apoferritin as a standard.
Venue: Journal of structural biology

Interpretation of the structural information in cryomicroscopy images recorded on film or CCD camera requires a precise knowledge of the electron microscope parameters that affect image features such as magnification and defocus. Magnification must be determined in order to combine data from different images in a three-dimensional reconstruction and to accurately scale reconstructions for fitting with atomic resolution models. A method is described for estimating the absolute magnification of an electron micrograph of a frozen-hydrated specimen using horse spleen apoferritin as a standard. Apoferritin is a widely available protein complex of known structure that may be included with the specimen of interest and imaged under conditions identical to those used for imaging other biological specimens by cryomicroscopy. The sum of the structure factor intensities of images of randomly-oriented apoferritin particles shows three low resolution peaks to 25Å that arise from the hollow ball structure of apoferritin. Comparison of peak positions of the experimental intensities with structure factor intensities of an atomic model of apoferritin determined by X-ray crystallography provides a scale factor for estimating the absolute magnification of the micrograph. We compare the magnification estimate using apoferritin to that obtained with tobacco mosaic virus, another common magnification standard for cryomicroscopy. We verify the precision of the method by acquiring images with a systematic variation of magnification.
Flavonoid interactions with human transthyretin: combined structural and thermodynamic analysis.
Venue: Journal of structural biology

Transthyretin (TTR) is a carrier protein involved in human amyloidosis. The development of small molecules that may act as TTR amyloid inhibitors is a promising strategy to treat these pathologies. Here we selected and characterized the interaction of flavonoids with the wild type and the V30M amyloidogenic mutant TTR. TTR acid aggregation was evaluated in vitro in the presence of the different flavonoids. The best TTR aggregation inhibitors were studied by Isothermal Titration Calorimetry (ITC) in order to reveal their thermodynamic signature of binding to TTRwt. Crystal structures of TTRwt in complex with the top binders were also obtained, enabling us to in depth inspect TTR interactions with these flavonoids. The results indicate that changing the number and position of hydroxyl groups attached to the flavonoid core strongly influence flavonoid recognition by TTR, either by changing ligand affinity or its mechanism of interaction with the two sites of TTR. We also compared the results obtained for TTRwt with the V30M mutant structure in the apo form, allowing us to pinpoint structural features that may facilitate or hamper ligand binding to the V30M mutant. Our data show that the TTRwt binding site is labile and, in particular, the central region of the cavity is sensible for the small differences in the ligands tested and can be influenced by the Met30 amyloidogenic mutation, therefore playing important roles in flavonoid binding affinity, mechanism and mutant protein ligand binding specificities.
Solution structure of an atypical PHD finger in BRPF2 and its interaction with DNA.
Venue: Journal of structural biology

Plant homeodomain (PHD) finger is found to be a versatile reader that functions in recruiting transcription factors and chromatin modification complexes. Bromodomain- and PHD finger-containing (BRPF) proteins are identified as scaffold component in a couple of histone acetyltransferase (HATs) complexes but the biological function of PHD fingers, composing the motif called PZPM (PHD/Zn-knuckle/PHD Motif), in BRPF proteins is far from being well understood. Here we report the three-dimensional solution structure of the second PHD finger of PZPM in human BRPF2. According to the structure, BRPF2 PHD2 possesses a two-strand β sheet which is different from any other PHD fingers. Functionally, this PHD finger can potentially bind DNA non-specifically with an evolutionarily conserved and positively charged surface. We provide the structural and interaction information of this atypical PHD finger and categorize this BRPF2 PHD2 into a new subset of PHD finger. Moreover our work also shed light on the functional aspect of the PZPM.
Contribution of hydrophobic interactions to the folding and fibrillation of histone H1 and its carboxy-terminal domain.
Venue: Journal of structural biology

Histone H1 is involved in chromatin structure and gene regulation. H1 also performs functions outside cell nuclei, which may depend on its properties as a lipid-binding protein. The H1 CTD behaves as an intrinsically disordered protein (IDP) with coupled binding and folding. Here, we used neutral detergents and anionic SDS to study the contribution of hydrophobic interactions to the folding of the CTD. In the presence of neutral detergents, the CTD folded with proportions of secondary structure motifs similar to those observed in the DNA complexes. These results identify a folding pathway for the CTD based on hydrophobic interactions, and independent of charge compensation. The CTD is phosphorylated to different extents by cyclin-dependent kinases. The general effect of phosphorylation in the presence of detergents was a decrease in the α-helix content and an increase in that of the β-structure. The greatest effect was observed in the fully phosphorylated CTD (three phosphate groups) in the presence of anionic SDS (7:1, detergent/CTD molar ratio); in these conditions, the CTD became an all-β protein, with 83% β-structure and no α-helix. The CTD in all-β conformation readily formed ribbon-like fibers. The entire H1 also formed fibers when fully phosphorylated in the CTD. Fibers were of the amyloid type, as judged by strong birefringence in the presence of Congo red and thioflavin fluorescence enhancement. Amyloid fiber formation was only observed in SDS, suggesting that it requires the joint effects of partial charge neutralization and hydrophobic interactions, together with the all-β potential provided by full phosphorylation.
Structural insights into decreased enzymatic activity induced by an insert sequence in mannonate dehydratase from Gram negative bacterium.
Venue: Journal of structural biology

Mannonate dehydratase (ManD; EC4.2.1.8) catalyzes the dehydration of D-mannonate to 2-keto-3-deoxygluconate. It is the third enzyme in the pathway for dissimilation of D-glucuronate to 2-keto-3-deoxygluconate involving in the Entner-Doudoroff pathway in certain bacterial and archaeal species. ManD from Gram negative bacteria has an insert sequence as compared to those from Gram positives revealed by sequence analysis. To evaluate the impact of this insert sequence on the catalytic efficiency, we solved the crystal structures of ManD from Escherichia coli strain K12 and its complex with D-mannonate, which reveal that this insert sequence forms two α helices locating above the active site. The two insert α helices introduce a loop that forms a cap covering the substrate binding pocket, which restricts the tunnels of substrate entering and product releasing from the active site. Site-directed mutations and enzymatic activity assays confirm that the catalytic rate is decreased by this loop. These features are conserved among Gram negative bacteria. Thus, the insert sequence of ManD from Gram negative bacteria acts as a common inducer to decrease the catalytic rate and consequently the glucuronate metabolic rate as compared to those from Gram positives. Moreover, residues essential for substrate to enter the active site were characterized via structural analysis and enzymatic activity assays.
3D structure determination of native mammalian cells using cryo-FIB and cryo-electron tomography.
Venue: Journal of structural biology

Cryo-electron tomography (cryo-ET) has enabled high resolution three-dimensional(3D) structural analysis of virus and host cell interactions and many cell signaling events; these studies, however, have largely been limited to very thin, peripheral regions of eukaryotic cells or to small prokaryotic cells. Recent efforts to make thin, vitreous sections using cryo-ultramicrotomy have been successful, however,this method is technically very challenging and with many artifacts. Here, we report a simple and robust method for creating in situ, frozen-hydrated cell lamellas using a focused ion beam at cryogenic temperature (cryo-FIB), allowing access to any interior cellular regions of interest. We demonstrate the utility of cryo-FIB with high resolution 3D cellular structures from both bacterial cells and large mammalian cells. The method will not only facilitate high-throughput 3D structural analysis of biological specimens, but is also broadly applicable to sample preparation of thin films and surface materials without the need for FIB "lift-out".
Pharmacological and structural characterization of conformationally restricted (S)-glutamate analogues at ionotropic glutamate receptors.
Venue: Journal of structural biology

Conformationally restricted glutamate analogues have been pharmacologically characterized at AMPA and kainate receptors and the crystal structures have been solved of the ligand (2S,1'R,2'S)-2-(2'-carboxycyclobutyl)glycine (CBG-IV) in complex with the ligand binding domains of the AMPA receptor GluA2 and the kainate receptor GluK3. These structures show that CBG-IV interacts with the binding pocket in the same way as (S)-glutamate. The binding affinities reveal that CBG-IV has high affinity at the AMPA and kainate receptor subtypes. Appreciable binding affinity of CBG-IV was not observed at NMDA receptors, where the introduction of the carbocyclic ring is expected to lead to a steric clash with binding site residues. CBG-IV was demonstrated to be an agonist at both GluA2 and the kainate receptor GluK1. CBG-IV showed high affinity binding to GluK1 compared to GluA2, GluK2 and GluK3, which exhibited lower affinity for CBG-IV. The structure of GluA2 LBD and GluK3 LBD in complex with CBG-IV revealed similar binding site interactions to those of (S)-glutamate. No major conformational rearrangements compared to the (S)-glutamate bound conformation were found in GluK3 in order to accommodate CBG-IV, in contrast with GluA2 where a shift in lobe D2 binding site residues occurs, leading to an increased binding cavity volume compared to the (S)-glutamate bound structure.
Structural delineation of histone post-translation modifications in histone-nucleosome assembly protein complex.
Venue: Journal of structural biology

Nucleosome assembly proteins (Nap) are histone chaperones with vital roles in chromatin assembly and disassembly. Decoding of histone post-translational modifications by histone chaperones is central in regulation of gene expression. We probed binding interfaces in Nap-histone complexes using histone peptide interaction arrays and chemical shift perturbation techniques. Phosphorylation at H3T32 and H3T118 enhanced Nap-peptide interactions while post-translational modifications like H3K122, H4K59 and H2AK36 diminished it. Nap recognition regions in histones H2A, H2B, H3 and H4 lie in core regions of the histone octamer, and form a surface exposed contiguous patch. Nap binding sites on H2A-H2B and H3-H4 fall on opposite surfaces of histone octamer, and the latter can therefore accommodate two dimeric Nap molecules simultaneously. Interfacial residues between Nap and histone octamer overlap with route of wrapped DNA on histone octamer, implying non-concurrent residency on the octamer by either Nap or DNA. Using NMR, we mapped Nap residues that undergo chemical shift perturbations in presence of histone peptides. Taken together, we provide an architectural model of a Nap-octamer complex which likely forms during chromatin reorganisation.
Identification and structural basis for a novel interaction between Vav2 and Arap3.
Venue: Journal of structural biology

Vav2 is a ubiquitous guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. It regulates processes including cell migration, neuronal development and phagocytosis through interactions with different proteins. In this study, Arap3, a dual GTPase-activating protein (GAP) for RhoA and Arf6, was first identified to be a novel interaction partner for Vav2 both in vitro and in vivo. ITC and NMR chemical shift perturbation experiments demonstrated that Vav2 SH2 domain was able to interact directly with phosphorylated Y1403 and Y1408 within the C-terminal region of Arap3 with high affinities, with the dissociation constants (Kd) of ≈ 0.27 and ≈ 1.40 μM, respectively. In addition, using different phosphotyrosine peptides, the pY +3 specificity of Vav2 SH2 domain was discovered. The solution structures of Vav2 SH2 domain in free and in complex with the phosphotyrosine peptide pY1408 were therefore determined to understand the structural basis of this recognition specificity. Structural analysis revealed that the presence of a Phe residue in the BG loop (BG6) leads to the formation of a shallow hydrophobic pY +3 pocket on the surface of Vav2 SH2 domain, which determines the pY +3 specificity of Vav2 SH2 domain.
Aggregation and fibril morphology of the Arctic mutation of Alzheimer's Aβ peptide by CD, TEM, STEM and in situ AFM.
Venue: Journal of structural biology

Morphology of aggregation intermediates, polymorphism of amyloid fibrils and aggregation kinetics of the "Arctic" mutant of the Alzheimer's amyloid β-peptide, Aβ((1-40))(E22G), in a physiologically relevant Tris buffer (pH 7.4) were thoroughly explored in comparison with the human wild type Alzheimer's amyloid peptide, wt-Aβ((1-40)), using both in situ atomic force and electron microscopy, circular dichroism and thioflavin T fluorescence assays. For arc-Aβ((1-40)) at the end of the 'lag'-period of fibrillization an abrupt appearance of ≈ 3 nm size 'spherical aggregates' with a homogeneous morphology, was identified. Then, the aggregation proceeds with a rapid growth of amyloid fibrils with a variety of morphologies, while the spherical aggregates eventually disappeared during in situ measurements. Arc-Aβ((1-40)) was also shown to form fibrils at much lower concentrations than wt-Aβ((1-40)): ≤ 2.5 μM and 12.5 μM, respectively. Moreover, at the same concentration, 50 μM, the aggregation process proceeds more rapidly for arc-Aβ((1-40)): the first amyloid fibrils were observed after c.a. 72 h from the onset of incubation as compared to approximately 7 days for wt-Aβ((1-40)). Amyloid fibrils of arc-Aβ((1-40)) exhibit a large variety of polymorphs, at least five, both coiled and non-coiled distinct fibril structures were recognized by AFM, while at least four types of arc-Aβ((1-40)) fibrils were identified by TEM and STEM and their mass-per-length statistics were collected suggesting supramolecular structures with two, four and six β-sheet laminae. Our results suggest a pathway of fibrillogenesis for full-length Alzheimer's peptides with small and structurally ordered transient spherical aggregates as on-pathway immediate precursors of amyloid fibrils.
A model for the structure of satellite tobacco mosaic virus.
Venue: Journal of structural biology

Satellite tobacco mosaic virus (STMV) is an icosahedral T=1 single-stranded RNA virus with a genome containing 1058 nucleotides. X-ray crystallography revealed a structure containing 30 double-helical RNA segments, with each helix having nine base pairs and an unpaired nucleotide at the 3' end of each strand. Based on this structure, Larson and McPherson proposed a model of 30 hairpin-loop elements occupying the edges of the icosahedron and connected by single-stranded regions. More recently, Schroeder et al. have combined the results of chemical probing with a novel helix searching algorithm to propose a specific secondary structure for the STMV genome, compatible with the Larson-McPherson model. Here we report an all-atom model of STMV, using the complete protein and RNA sequences and the Schroeder RNA secondary structure. As far as we know, this is the first all-atom model for the complete structure of any virus (100% of the atoms) using the natural genomic sequence.
TxBR montage reconstruction for large field electron tomography.
Venue: Journal of structural biology

Electron tomography (ET) has been proven an essential technique for imaging the structure of cells beyond the range of the light microscope down to the molecular level. Large-field high-resolution views of biological specimens span more than four orders of magnitude in spatial scale, and, as a consequence, are rather difficult to generate directly. Various techniques have been developed towards generating those views, from increasing the sensor array size to implementing serial sectioning and montaging. Datasets and reconstructions obtained by the latter techniques generate multiple three-dimensional (3D) reconstructions, that need to be combined together to provide all the multiscale information. In this work, we show how to implement montages within TxBR, a tomographic reconstruction software package. This work involves some new application of mathematical concepts related to volume preserving transformations and issues of gauge ambiguity, which are essential problems arising from the nature of the observation in an electron microscope. The purpose of TxBR is to handle those issues as generally as possible in order to correct for most distortions in the 3D reconstructions and allow for a seamless recombination of ET montages.
CDC-48/p97 is required for proper meiotic chromosome segregation via controlling AIR-2/Aurora B kinase localization in Caenorhabditis elegans.
Venue: Journal of structural biology

CDC-48/p97 is a AAA (ATPases associated with diverse cellular activities) chaperone involved in protein conformational changes such as the disassembly of protein complexes. We previously reported that Caenorhabditis elegans CDC-48.1 and CDC-48.2 (CDC-48s) are essential for the progression of meiosis I metaphase. Here, we report that CDC-48s are required for proper chromosome segregation during meiosis in C. elegans. In wild-type worms, at the diakinesis phase, phosphorylation of histone H3, one of the known substrates of aurora B kinase (AIR-2), on meiosis I chromatids correlated with AIR-2 localization at the cohesion sites of homologous chromatids. Conversely, depletion of CDC-48s resulted in a significant expansion of signals for AIR-2 and phosphorylated histone H3 over the entire length of meiotic chromosomes, leading to defective chromosome segregation, while the total amount of AIR-2 in lysates was not changed by the depletion of CDC-48s. The defective segregation of meiotic chromosomes caused by the depletion of CDC-48s was suppressed by the simultaneous depletion of AIR-2 and is similar to that observed following the depletion of protein phosphatase 1 (PP1) phosphatases. However, the amount and localization of PP1 were not changed by the depletion of CDC-48s. These results suggest that CDC-48s control the restricted localization of AIR-2 to the cohesion sites of homologous chromatids in meiosis I.
Pores of the toxin FraC assemble into 2D hexagonal clusters in both crystal structures and model membranes.
Venue: Journal of structural biology

The recent high-resolution structure of the toxin FraC derived from the sea anemone Actinia fragacea has provided new insight into the mechanism of pore formation by actinoporins. In this work, we report two new crystal forms of FraC in its oligomeric prepore conformation. Together with the previously reported structure, these two new structures reveal that ring-like nonamers of the toxin assemble into compact two-dimensional hexagonal arrays. This supramolecular organization is maintained in different relative orientations adopted by the oligomers within the crystal layers. Analyses of the aggregation of FraC pores in both planar and curved (vesicles) model membranes show similar 2D hexagonal arrangements. Our observations support a model in which hexagonal pore-packing is a clustering mechanism that maximizes toxin-driven membrane damage in the target cell.