nils-herrmann/clean_scirepeval huggingface dataset:
goCTF: Geometrically optimized CTF determination for single-particle cryo-EM.
Venue: Journal of structural biology

Preferred particle orientation represents a recurring problem in single-particle cryogenic electron microcopy (cryo-EM). A specimen-independent approach through tilting has been attempted to increase particle orientation coverage, thus minimizing anisotropic three-dimensional (3D) reconstruction. However, focus gradient is a critical issue hindering tilt applications from being a general practice in single-particle cryo-EM. The present study describes a newly developed geometrically optimized approach, goCTF, to reliably determine the global focus gradient. A novel strategy of determining contrast transfer function (CTF) parameters from a sector of the signal preserved power spectrum is applied to increase reliability. Subsequently, per-particle based local focus refinement is conducted in an iterative manner to further improve the defocus accuracy. Novel diagnosis methods using a standard deviation defocus plot and goodness of fit heatmap have also been proposed to evaluate CTF fitting quality prior to 3D refinement. In a benchmark study, goCTF processed a published single-particle cryo-EM dataset for influenza hemagglutinin trimer collected at a 40-degree specimen tilt. The resulting 3D reconstruction map was improved from 4.1 Å to 3.7 Å resolution. The goCTF program is built on the open-source code of CTFFIND4, which adopts a consistent user interface for ease of use.
Pig enamel revisited - Incremental markings in enamel of wild boars and domestic pigs.
Venue: Journal of structural biology

The nature and periodicity of incremental markings in pig enamel is currently debated. To broaden the basis for a correct interpretation of growth marks in pig enamel, we analyzed their periodicity in teeth of wild boars and domestic pigs. For that, the numbers of enamel incremental markings were recorded in ground sections and compared with crown formation times for the respective teeth derived from literature data on tooth development and eruption in Sus scrofa. Our study revealed that laminations with a daily periodicity are the dominant incremental feature of pig enamel. In wild boar M3s, daily enamel secretion (apposition) rates ranged between a minimum of 6.1 µm in the inner and a maximum of 30.6 µm in the outer enamel. Long-period (supra-daily) incremental markings were present as perikymata at the outer enamel surface (OES). Contrary to the situation in primate enamel, in pig enamel the long-period incremental lines terminating in perikyma grooves were mostly structurally indistinguishable from the daily laminations. Typically, five sub-daily increments were present between successive laminations. The incremental pattern in pig enamel can be misinterpreted if the laminations are mistaken for long-period markings (striae of Retzius) and the sub-daily growth marks for daily prism cross-striations. The findings of the present study demonstrate the critical importance of correctly characterizing the incremental markings and their periodicity in enamel, and caution against an uncritical transfer of the interpretation of the nature of incremental markings in primate enamel to other mammalian taxa.
Variation in human hair ultrastructure among three biogeographic populations.
Venue: Journal of structural biology

Human scalp hairs are often examined microscopically to study the variation and diversity among a range of visible morphological traits. In this study, we focused on the ultrastructure of human scalp hair within its keratinized matrix, emphasizing, the density and distribution of melanosomes, variation in cuticle thickness within populations, and the relationship of hair fiber ultrastructure with biogeographic ancestry. We used transmission electron microscopy (TEM) to visualize hair cross-sections and generate micron-scale resolution images for analysis of particle morphology and the layered hair matrix. Our results revealed considerable variation in all parameters examined, including the relationship of ultrastructure to biogeographic ancestry. Among the three metapopulations studied (European, African, and East Asian), we identified hair cross-sectional shape, cuticle dimensions, and melanosome distribution as traits that reveal statistically significant ancestry-related patterns. This study establishes trait patterns in hair morphology and ultrastructure among three biogeographically defined metapopulations to improve the current understanding of human variation in hair form and establish a foundation for future studies on the genetic and developmental bases of phenotypic variation in hair ultrastructure related to genotype.
Ex vivo detection of calcium phosphate and calcium carbonate in rat blood serum.
Venue: Journal of structural biology

The total calcium (tCa) in blood serum comprises free Ca2+ ions (fCa), protein-bound calcium (prCa), and complexed calcium by small anions (cCa). The cCa fraction, in addition to fCa, has been indicated to have some physiological activity. However, there is little evidence for the structure of its constituents. Here we report an ex vivo detection of the cCa constituents by synchrotron X-ray absorption near-edge structure spectroscopy. We collected the data directly on rat blood serum and, by making use of the reference samples, derived a spectrum that exhibits the features of cCa constituents. Among the features are those of the complexes of calcium phosphate and calcium carbonate. The detected complexes in the cCa fraction are mainly Ca(η2-HPO4)(H2O)4 and Ca(η1-HCO3)(H2O)5+, in which HPO42- and HCO3- serve as bidentate and unidentate ligands, respectively. The remained H2O molecules on the coordination sphere of Ca2+ enable these complexes to behave partially like aquated Ca2+ ions in protein-binding. Besides, as the dominant part of prCa, albumin-bound calcium (albCa) exhibits a spectrum that closely resembles that of fCa, indicating weak interactions between the protein carboxyl groups and calcium. The weak-bound cCa and albCa, along with fCa and the relevant anions, compose a local chemical system that could play a role in maintaining the calcium level in blood.
Structural insights into the catalytic mechanism of 5-methylthioribose 1-phosphate isomerase.
Venue: Journal of structural biology

5-Methylthioribose 1-phosphate isomerase (M1Pi) is a crucial enzyme involved in the universally conserved methionine salvage pathway (MSP) where it is known to catalyze the conversion of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P) via a mechanism which remains unspecified till date. Furthermore, although M1Pi has a discrete function, it surprisingly shares high structural similarity with two functionally non-related proteins such as ribose-1,5-bisphosphate isomerase (R15Pi) and the regulatory subunits of eukaryotic translation initiation factor 2B (eIF2B). To identify the distinct structural features that lead to divergent functional obligations of M1Pi as well as to understand the mechanism of enzyme catalysis, the crystal structure of M1Pi from a hyperthermophilic archaeon Pyrococcus horikoshii OT3 was determined. A meticulous structural investigation of the dimeric M1Pi revealed the presence of an N-terminal extension and a hydrophobic patch absent in R15Pi and the regulatory α-subunit of eIF2B. Furthermore, unlike R15Pi in which a kink formation is observed in one of the helices, the domain movement of M1Pi is distinguished by a forward shift in a loop covering the active-site pocket. All these structural attributes contribute towards a hydrophobic microenvironment in the vicinity of the active site of the enzyme making it favorable for the reaction mechanism to commence. Thus, a hydrophobic active-site microenvironment in addition to the availability of optimal amino-acid residues surrounding the catalytic residues in M1Pi led us to propose its probable reaction mechanism via a cis-phosphoenolate intermediate formation.
Changing times: Fluorescence-lifetime analysis of amyloidogenic SF-IAPP fusion protein.
Venue: Journal of structural biology

In a number of conformational diseases, intracellular accumulation of proteins bearing non-native conformations occurs. The search for compounds that are capable of hindering the formation and accumulation of toxic protein aggregates and fibrils is an urgent task. Present fluorescent methods of fibrils' detection prevent simple real-time observations. We suppose to use green fluorescent protein fused with target protein and fluorescence lifetime measurement technique for this purpose. The recombinant proteins analyzed were produced in E. coli. Mass spectrometry was used for the primary structure of the recombinant proteins and post-translational modifications identification. The fluorescence lifetime of the superfolder green fluorescent protein (SF) and the SF protein fused with islet amyloid polypeptide (SF-IAPP) were studied in polyacrylamide gel using Fluorescent-Lifetime Imaging Microscopy (FLIM). It was shown that the SF average fluorescence lifetime in gel slightly differs from that of the SF-IAPP monomer under these conditions. SF-IAPP does not lose the ability to form amyloid-like fibrils. Under the same conditions (in polyacrylamide gel), SF and SF-IAPP monomers have similar fluorescence time characteristics and the average fluorescence lifetime of SF-IAPP in fibrils significantly decreases. We propose the application of FLIM to the measurement of average fluorescence lifetimes of fusion proteins (amyloidogenic protein-SF) in the context of studies using cellular models of conformational diseases.
Analysis of a new flavodiiron core structural arrangement in Flv1-ΔFlR protein from Synechocystis sp. PCC6803.
Venue: Journal of structural biology

Flavodiiron proteins (FDPs) play key roles in biological response mechanisms against oxygen and/or nitric oxide; in particular they are present in oxygenic phototrophs (including cyanobacteria and gymnosperms). Two conserved domains define the core of this family of proteins: a N-terminal metallo-β-lactamase-like domain followed by a C-terminal flavodoxin-like one, containing the catalytic diiron centre and a FMN cofactor, respectively. Members of the FDP family may present extra modules in the C-terminus, and were classified into several classes according to their distribution and composition. The cyanobacterium Synechocystis sp. PCC6803 contains four Class C FDPs (Flv1-4) that include at the C-terminus an additional NAD(P)H:flavin oxidoreductase (FlR) domain. Two of them (Flv3 and Flv4) have the canonical diiron ligands (Class C, Type 1), while the other two (Flv1 and Flv2) present different residues in that region (Class C, Type 2). Most phototrophs, either Bacterial or Eukaryal, contain at least two FDP genes, each encoding for one of those two types. Crystals of the Flv1 two core domains (Flv1-ΔFlR), without the C-terminal NAD(P)H:flavin oxidoreductase extension, were obtained and the structure was determined. Its pseudo diiron site contains non-canonical basic and neutral residues, and showed anion moieties, instead. The presented structure revealed for the first time the structure of the two-domain core of a Class C-Type 2 FDP.
Crystal structure and substrate recognition mechanism of Aspergillus oryzae isoprimeverose-producing enzyme.
Venue: Journal of structural biology

Isoprimeverose-producing enzymes (IPases) release isoprimeverose (α-d-xylopyranosyl-(1 → 6)-d-glucopyranose) from the non-reducing end of xyloglucan oligosaccharides. Aspergillus oryzae IPase (IpeA) is classified as a member of the glycoside hydrolase family 3 (GH3); however, it has unusual substrate specificity compared with other GH3 enzymes. Xylopyranosyl branching at the non-reducing ends of xyloglucan oligosaccharides is vital for IpeA activity. We solved the crystal structure of IpeA with isoprimeverose at 2.4 Å resolution, showing that the structure of IpeA formed a dimer and was composed of three domains: an N-terminal (β/α)8 TIM-barrel domain, α/β/α sandwich fold domain, and a C-terminal fibronectin-like domain. The catalytic TIM-barrel domain possessed a catalytic nucleophile (Asp300) and acid/base (Glu524) residues. Interestingly, we found that the cavity of the active site of IpeA was larger than that of other GH3 enzymes, and subsite -1' played an important role in its activity. The glucopyranosyl and xylopyranosyl residues of isoprimeverose were located at subsites -1 and -1', respectively. Gln58 and Tyr89 contributed to the interaction with the xylopyranosyl residue of isoprimeverose through hydrogen bonding and stacking effects, respectively. Our findings provide new insights into the substrate recognition of GH3 enzymes.
Structural and biochemical characterization of citrate binding to AtPPC3, a plant-type phosphoenolpyruvate carboxylase from Arabidopsis thaliana.
Venue: Journal of structural biology

Phosphoenolpyruvate carboxylase (PEPC) is a tightly regulated cytosolic enzyme situated at a crucial branch point of central plant metabolism. The structure of AtPPC3, a C3 PEPC isozyme of the model plant Arabidopsis thaliana, in complex with the inhibitors aspartate and citrate was solved at 2.2-Å resolution. This represents the first PEPC structure with citrate bound. Aspartate and citrate binding sites are in close proximity (5.1-5.3 Å) and interactions between citrate and specific residues were identified. Citrate functions as a mixed (allosteric) inhibitor as it reduced AtPPC3's Vmax while increasing Km(PEP) values. The PEP saturation data gave an excellent fit to the mixed inhibition model, yielding Ki and Ki' (citrate) values of 9.3 and 42.5 mM, respectively. Citrate and aspartate inhibition of AtPPC3 was non-additive, likely due to their closely positioned binding sites, their similar negative charge, and type of binding residues. Fewer interactions and lower affinity for citrate support its observed weaker inhibition of AtPPC3 relative to aspartate. Citrate does not appear to induce further conformational change beyond aspartate owing to the similar structural mechanism of inhibition. AtPPC3 largely exhibits root-specific expression in Arabidopsis, where it is markedly upregulated during stresses such as excessive salinity or nutritional Pi deprivation that necessitate large increases in anaplerotic PEP carboxylation. The cytosolic citrate concentration of potato tubers suggests that AtPPC3's inhibition by citrate may be physiologically relevant. Our results provide novel insights into the structural basis of allosteric PEPC control and the kinetic effects brought about upon inhibitor binding.
Structural dissection of sterol glycosyltransferase UGT51 from Saccharomyces cerevisiae for substrate specificity.
Venue: Journal of structural biology

Sterol glycosyltransferases catalyze the formation of a variety of glycosylated sterol derivatives and are involved in producing a plethora of bioactive natural products. To understand the molecular mechanism of sterol glycosyltransferases, we determined crystal structures of a sterol glycosyltransferase UGT51 from Saccharomyces cerevisiae. The structures of the UGT51 and its complex with uridine diphosphate glucose (UDPG) were solved at resolutions of 2.77 Å and 1.9 Å, respectively. The structural analysis revealed that a long hydrophobic cavity, 9.2 Å in width and 17.6 Å in length located at the N-terminal domain of UGT51, is suitable for the accommodation of sterol acceptor substrates. Furthermore, a short, conserved sequence of S847-M851 was identified at the bottom of the hydrophobic cavity, which might be the steroid binding site and play an important role for the UGT51 catalytic specificity towards sterols. Molecular docking simulations indicated that changed unique interaction network in mutant M7_1 (S801A/L802A/V804A/K812A/E816K/S849A/N892D), with an 1800-fold activity improvement toward an unnatural substrate protopanaxadiol (PPD), might influence its substrate preference. This study reported the first sterol glycosyltransferase structure, providing a molecular blueprint for generating tailored sterol glycosyltransferases as potential catalytic elements in synthetic biology.
Structural and biochemical analysis of a NOT1 MIF4G-like domain of the CCR4-NOT complex.
Venue: Journal of structural biology

The CCR4-NOT complex plays a central role in the regulation of gene expression and degradation of messenger RNAs. The multisubunit complex assembles on the NOT1 protein, which acts as a 'scaffold' and is highly conserved in eukaryotes. NOT1 consists of a series of helical domains that serve as docking sites for other CCR4-NOT subunits. We describe a crystal structure of a connector domain of NOT1 from the thermophilic fungus Chaetomium thermophilum (Ct). Comparative structural analysis indicates that this domain adopts a MIF4G-like fold and we have termed it the MIF4G-C domain. Solution scattering studies indicate that the human MIF4G-C domain likely adopts a very similar fold to the Ct MIF4G-C. MIF4G domains have been described to mediate interactions with DEAD-box helicases such as DDX6. However, comparison of the interfaces of the MIF4G-C with the MIF4G domain of NOT1 that interacts with DDX6 reveals key structural differences that explain why the MIF4G-C does not bind DDX6. We further show that the human MIF4G-C does not interact stably with other subunits of the CCR4-NOT complex. The structural conservation of the MIF4G-C domain suggests that it may have an important but presently undefined role in the CCR4-NOT complex.
Crystal structures and biochemical characterization of DNA sliding clamps from three Gram-negative bacterial pathogens.
Venue: Journal of structural biology

Bacterial sliding clamps bind to DNA and act as protein-protein interaction hubs for several proteins involved in DNA replication and repair. The partner proteins all bind to a common pocket on sliding clamps via conserved linear peptide sequence motifs, which suggest the pocket as an attractive target for development of new antibiotics. Herein we report the X-ray crystal structures and biochemical characterization of β sliding clamps from the Gram-negative pathogens Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacter cloacae. The structures reveal close similarity between the pathogen and Escherichia coli clamps and similar patterns of binding to linear clamp-binding motif peptides. The results suggest that linear motif-sliding clamp interactions are well conserved and an antibiotic targeting the sliding clamp should have broad-spectrum activity against Gram-negative pathogens.
Investigating HCMV entry into host cells by STEM tomography.
Venue: Journal of structural biology

Human cytomegalovirus (HCMV) entry into susceptible cells is a fast intricate process that is not fully understood. Although, previous studies explored different aspects of this process by means of biochemical and inhibitors assays, a clear morphological characterization of its steps at the ultrastructural level is still lacking. We attempted to characterize those intermediates involved during HCMV entry by developing a methodological approach that resulted in optimal ultrastructure preservation and allowed for 3D imaging. It involves rapid freezing and cryosubstitution which ensure a clear visibility of membranous leaflets as well as retained membranous continuity. Likewise, it delivered a reproducible optimization of the growth and infection conditions that are pivotal towards maintaining biologically active enriched input virus particles. Data acquisition was achieved through STEM tomography in a 3D context. Indeed, several intermediates that characterize HCMV entry-related events were observed both extra- and intracellularly. Some of the cell-membrane associated viral particles that we referred to as "Pinocchio particles" were morphologically altered in comparison to the cell-free virions. We were also able to characterize intracellular fusion intermediates taking place between the viral envelope and the vesicular membranes. Furthermore, inhibiting actin polymerization by Latrunculin-A enabled us to spot fusion-like intermediates of the viral envelope with the host cell plasma membrane that we did not observe in the untreated infected cells. Our data also suggests that Dyngo-4a; a dynamin-2 inhibitor, does not interfere with the internalization of the HCMV into the host cells as previously deduced.
Structure and function of Mycobacterium-specific components of F-ATP synthase subunits α and ε.
Venue: Journal of structural biology

The Mycobacterium tuberculosis (Mtb) F1FO-ATP synthase (α3:β3:γ:δ:ε:a:b:b':c9) is an essential enzyme that supplies energy for both the aerobic growing and the hypoxic dormant stage of the mycobacterial life cycle. Employing the heterologous F-ATP synthase model system αchi3:β3:γ we showed previously, that transfer of the C-terminal domain (CTD) of Mtb subunit α (Mtα514-549) to a standard F-ATP synthase α subunit suppresses ATPase activity. Here we determined the 3D reconstruction from electron micrographs of the αchi3:β3:γ complex reconstituted with the Mtb subunit ε (Mtε), which has been shown to crosstalk with the CTD of Mtα. Together with the first solution shape of Mtb subunit α (Mtα), derived from solution X-ray scattering, the structural data visualize the extended C-terminal stretch of the mycobacterial subunit α. In addition, Mtε mutants MtεR62L, MtεE87A, Mtε6-121, and Mtε1-120, reconstituted with αchi3:β3:γ provided insight into their role in coupling and in trapping inhibiting MgADP. NMR solution studies of MtεE87A gave insights into how this residue contributes to stability and crosstalk between the N-terminal domain (NTD) and the CTD of Mtε. Analyses of the N-terminal mutant Mtε6-121 highlight the differences of the NTD of mycobacterial subunit ε to the well described Geobacillus stearothermophilus or Escherichia coli counterparts. These data are discussed in context of a crosstalk between the very N-terminal amino acids of Mtε and the loop region of one c subunit of the c-ring turbine for coupling of proton-translocation and ATP synthesis activity.
The structure of DcrB, a lipoprotein from Salmonella enterica, reveals flexibility in the N-terminal segment of the Mog1p/PsbP-like fold.
Venue: Journal of structural biology

DcrB is an 18 kDa lipoprotein that contains a single domain of unknown function. DcrB is found within Enterobacteriaceae, a family of Gram-negative bacteria which includes pathogens that can cause food-borne illness and hospital-acquired infections. In Salmonella enterica serovar Typhimurium, DcrB is up-regulated by conditions that promote the production of known virulence factors. We determined the structure of a truncated form of DcrB from Salmonella to 1.92 Å resolution by X-ray crystallography. This truncated form, DcrBΔ37, contains the entire domain of unknown function but lacks the lipoprotein signal sequence (residues 1-20) as well as residues 21-37. The DcrBΔ37 monomer contains the Mog1p/PsbP-like fold, which is found in functionally diverse proteins in mammals, yeast, plants, and cyanobacteria. Interestingly, DcrBΔ37 crystallized as a domain-swapped homodimer in which the N-terminal β-hairpin extends from one protomer to interact with the core of the second protomer. This domain-swapping indicates that the N-terminal portion of the Mog1p/PsbP-like fold likely has conformational flexibility. Overall, our results provide the first example of an enterobacterial protein that contains the Mog1p/PsbP-like fold and expands knowledge of the structural and phylogenetic diversity of Mog1p/PsbP-like proteins.