nils-herrmann/clean_scirepeval huggingface dataset:
X-ray mosaic nanotomography of large microorganisms.
Venue: Journal of structural biology

Full-field X-ray microscopy is a valuable tool for 3D observation of biological systems. In the soft X-ray domain organelles can be visualized in individual cells while hard X-ray microscopes excel in imaging of larger complex biological tissue. The field of view of these instruments is typically 10(3) times the spatial resolution. We exploit the assets of the hard X-ray sub-micrometer imaging and extend the standard approach by widening the effective field of view to match the size of the sample. We show that global tomography of biological systems exceeding several times the field of view is feasible also at the nanoscale with moderate radiation dose. We address the performance issues and limitations of the TOMCAT full-field microscope and more generally for Zernike phase contrast imaging. Two biologically relevant systems were investigated. The first being the largest known bacteria (Thiomargarita namibiensis), the second is a small myriapod species (Pauropoda sp.). Both examples illustrate the capacity of the unique, structured condenser based broad-band full-field microscope to access the 3D structural details of biological systems at the nanoscale while avoiding complicated sample preparation, or even keeping the sample environment close to the natural state.
Super-resolution fluorescence imaging of chromosomal DNA.
Venue: Journal of structural biology

Super-resolution microscopy is a powerful tool for understanding cellular function. However one of the most important biomolecules - DNA - remains somewhat inaccessible because it cannot be effectively and appropriately labeled. Here, we demonstrate that robust and detailed super-resolution images of DNA can be produced by combining 5-ethynyl-2'-deoxyuridine (EdU) labeling using the 'click chemistry' approach and direct stochastic optical reconstruction microscopy (dSTORM). This method can resolve fine chromatin structure, and - when used in conjunction with pulse labeling - can reveal the paths taken by individual fibers through the nucleus. This technique should provide a useful tool for the study of nuclear structure and function.
Insights from the architecture of the bacterial transcription apparatus.
Venue: Journal of structural biology

We provide a portrait of the bacterial transcription apparatus in light of the data emerging from structural studies, sequence analysis and comparative genomics to bring out important but underappreciated features. We first describe the key structural highlights and evolutionary implications emerging from comparison of the cellular RNA polymerase subunits with the RNA-dependent RNA polymerase involved in RNAi in eukaryotes and their homologs from newly identified bacterial selfish elements. We describe some previously unnoticed domains and the possible evolutionary stages leading to the RNA polymerases of extant life forms. We then present the case for the ancient orthology of the basal transcription factors, the sigma factor and TFIIB, in the bacterial and the archaeo-eukaryotic lineages. We also present a synopsis of the structural and architectural taxonomy of specific transcription factors and their genome-scale demography. In this context, we present certain notable deviations from the otherwise invariant proteome-wide trends in transcription factor distribution and use it to predict the presence of an unusual lineage-specifically expanded signaling system in certain firmicutes like Paenibacillus. We then discuss the intersection between functional properties of transcription factors and the organization of transcriptional networks. Finally, we present some of the interesting evolutionary conundrums posed by our newly gained understanding of the bacterial transcription apparatus and potential areas for future explorations.
Correlative VIS-fluorescence and soft X-ray cryo-microscopy/tomography of adherent cells
Venue: Journal of structural biology

Soft X-ray cryo-microscopy/tomography of vitreous samples is becoming a valuable tool in structural cell biology. Within the ‘water-window’ wavelength region (2.34–4.37 nm), it provides absorption contrast images with high signal to noise ratio and resolution of a few tens of nanometer. Soft X-rays with wavelengths close to the K-absorption edge of oxygen penetrate biological samples with thicknesses in the micrometer range. Here, we report on the application of a recently established extension of the transmission soft X-ray cryo-microscope (HZB TXM) at the beamline U41-XM of the BESSY II electron storage ring by an in-column epi-fluorescence and reflected light cryo-microscope. We demonstrate the new capability for correlative fluorescence and soft X-ray cryo-microscopy/tomography of this instrument along a typical life science experimental approach – the correlation of a fluorophore-tagged protein (pUL34-GFP of pseudorabies virus, PrV, the nuclear membrane-anchored component of the nuclear egress complex of the Herpesviridae which interacts with viral pUL31) in PrV pUL34-GFP/pUL31 coexpressing mammalian cells, with virus-induced vesicular structures in the nucleus, expanding the nucleoplasmic reticulum. Taken together, our results demonstrate new possibilities to study the role of specific proteins in substructures of adherent cells, especially of the nucleus in toto, accessible to electron microscopy in thinned samples only.
Crystal structure of Sa239 reveals the structural basis for the activation of ribokinase by monovalent cations.
Venue: Journal of structural biology

Ribokinase is responsible for catalyzing the reaction of d-ribose and ATP to produce ribose-5-phosphate and ADP, which can be activated by monovalent cations such as potassium, cesium and ammonium. However, the exact activation mechanism of ribokinase remains elusive. Here we report the crystal structure of Sa239, a ribokinase from Staphylococcus aureus, in the absence of monovalent ions. In addition to the dimer form similar to that observed in Escherichia coli ribokinase structure, the structure of Sa239 demonstrates that the C-terminal tail protrudes from the remaining part and interacts with the neighboring molecule, resulting in an unexpected dimerization form. By comparing the structure of Sa239 to E. coli ribokinase, we propose that binding of the monovalent cation triggers the conformational change of the large ATP loop to organize the formation of nucleotide binding pocket, thus enabling ATP binding and enhancing catalytic activity. Our study uncovers the detailed structural basis for the activation mechanism of ribokinase by monovalent cations.
Water's potential role: Insights from studies of the p53 core domain.
Venue: Journal of structural biology

Soluble proteins with amyloidogenic propensity such as the tumor suppressor protein p53 have high proportion of incompletely desolvated backbone H bonds (HB). Such bonds are vulnerable to water attack, thus potentially leading to the misfolding of these proteins. However, it is still not clear how the surrounding solvent influences the protein native states. To address this, systematic surveys by molecular dynamics simulations and entropy analysis were performed on the p53 core domain in this work. We examined seven wild/mutant X-ray structures and observed two types of water-network hydration in three "hot hydration centers" (DNA- or small molecule- binding surfaces of the p53 core domain). The "tight" water, resulting from the local collective hydrogen-bond interactions, is probably fundamental to the protein structural stability. The second type of water is highly "dynamical" and exchanges very fast within the bulk solution, which is unambiguously assisted by the local protein motions. An entropy mapping of the solvent around the protein and a temperature perturbation analysis further present the main features of the p53 hydration network. The particular environment created by different water molecules around the p53 core domain also partly explains the structural vulnerabilities of this protein.
Nanoindentation characterization of the micro-lamellar arrangement of black coral skeleton.
Venue: Journal of structural biology

Black corals (antipatharians) are colonial cnidarians whose branched tree-like skeleton is mostly constituted of chitin fibrils inside a lipoproteic matrix. The skeleton exhibits growth rings formed by chitin layers (micro-lamellae). In order to know the effect of the arrangement microlamellae of chitin of black corals and to improve the understanding of the role of chitin structure in the antipatharian skeleton, the mechanical properties of the skeleton of two black corals, Antipathes caribbeana and Antipathes pennacea, were examined using nanoindentation tests. Measurements of reduced elastic modulus, nanohardness and the viscoelastic behavior were measured with a spheroconical indenter. The results indicate variations in the values of the mechanical properties clearly associated with different structures present in the skeletons, the core being the one that invariably shows the maximum values. The solid multilamellar arrangement of black coral chitin, its viscoelastic behavior, and the anisotropic mechanical response, are relevant factors contributing to the successful adaptation of black coral colonies to shallow as well as to very deep waters.
PyTom: a python-based toolbox for localization of macromolecules in cryo-electron tomograms and subtomogram analysis.
Venue: Journal of structural biology

Cryo-electron tomography (CET) is a three-dimensional imaging technique for structural studies of macromolecules under close-to-native conditions. In-depth analysis of macromolecule populations depicted in tomograms requires identification of subtomograms corresponding to putative particles, averaging of subtomograms to enhance their signal, and classification to capture the structural variations among them. Here, we introduce the open-source platform PyTom that unifies standard tomogram processing steps in a python toolbox. For subtomogram averaging, we implemented an adaptive adjustment of scoring and sampling that clearly improves the resolution of averages compared to static strategies. Furthermore, we present a novel stochastic classification method that yields significantly more accurate classification results than two deterministic approaches in simulations. We demonstrate that the PyTom workflow yields faithful results for alignment and classification of simulated and experimental subtomograms of ribosomes and GroEL(14)/GroEL(14)GroES(7), respectively, as well as for the analysis of ribosomal 60S subunits in yeast cell lysate. PyTom enables parallelized processing of large numbers of tomograms, but also provides a convenient, sustainable environment for algorithmic development.
Structural basis of specificity in tetrameric Kluyveromyces lactis β-galactosidase.
Venue: Journal of structural biology

β-Galactosidase or lactase is a very important enzyme in the food industry, being that from the yeast Kluyveromyces lactis the most widely used. Here we report its three-dimensional structure both in the free state and complexed with the product galactose. The monomer folds into five domains in a pattern conserved with the prokaryote enzymes of the GH2 family, although two long insertions in domains 2 and 3 are unique and related to oligomerization and specificity. The tetrameric enzyme is a dimer of dimers, with higher dissociation energy for the dimers than for its assembly. Two active centers are located at the interface within each dimer in a narrow channel. The insertion at domain 3 protrudes into this channel and makes putative links with the aglycone moiety of docked lactose. In spite of common structural features related to function, the determinants of the reaction mechanism proposed for Escherichia coli β-galactosidase are not found in the active site of the K. lactis enzyme. This is the first X-ray crystal structure for a β-galactosidase used in food processing.
Structural and functional analysis of the flexible regions of the catalytic α-subunit of protein kinase CK2.
Venue: Journal of structural biology

CK2 is a Ser/Thr protein kinase essential for cell viability. Its activity is anomalously high in several solid (prostate, mammary gland, lung, kidney and head and neck) and haematological tumours (AML, CML and PML), creating conditions favouring the onset of cancer. Cancer cells become addicted to high levels of CK2 activity and therefore this kinase is a remarkable example of "non-oncogene addiction". CK2 is a validated target for cancer therapy with one inhibitor in phase I clinical trials. Several crystal structures of CK2 are available, many in complex with ATP-competitive inhibitors, showing the presence of regions with remarkable flexibility. We present the structural characterisation of these regions by means of seven new crystal structures, in the apo form and in complex with inhibitors. We confirm previous findings about the unique flexibility of the CK2α catalytic subunit in the hinge/αD region, the p-loop and the β4β5 loop, and show here that there is no clear-cut correlation between the conformations of these flexible zones. Our findings challenge some of the current interpretations on the functional role of these regions and dispute the hypothesis that small ligands stabilize an inactive state. The mobility of the hinge/αD region in the human enzyme is unique among protein kinases, and this can be exploited for the development of more selective ATP-competitive inhibitors. The identification of different ligand binding modes to a secondary site can provide hints for the design of non-ATP-competitive inhibitors targeting the interaction between the α catalytic and the β regulatory subunits.
Detection of secondary and supersecondary structures of proteins from cryo-electron microscopy.
Venue: Journal of structural biology

Recent advances in three-dimensional electron microscopy (3D EM) have enabled the quantitative visualization of the structural building blocks of proteins at improved resolutions. We provide algorithms to detect the secondary structures (α-helices and β-sheets) from proteins for which the volumetric maps are reconstructed at 6-10Å resolution. Additionally, we show that when the resolution is coarser than 10Å, some of the supersecondary structures can be detected from 3D EM maps. For both these algorithms, we employ tools from computational geometry and differential topology, specifically the computation of stable/unstable manifolds of certain critical points of the distance function induced by the molecular surface. Our results connect mathematically well-defined constructions with bio-chemically induced structures observed in proteins.
Biomedical applications of the ESRF synchrotron-based microspectroscopy platform.
Venue: Journal of structural biology

Very little is known about the sub-cellular distribution of metal ions in cells. Some metals such as zinc, copper and iron are essential and play an important role in the cell metabolism. Dysfunctions in this delicate housekeeping may be at the origin of major diseases. There is also a prevalent use of metals in a wide range of diagnostic agents and drugs for the diagnosis or treatment of a variety of disorders. This is becoming more and more of a concern in the field of nanomedicine with the increasing development and use of nanoparticles, which are suspected of causing adverse effects on cells and organ tissues. Synchrotron-based X-ray and Fourier-transformed infrared microspectroscopies are developing into well-suited sub-micrometer analytical tools for addressing new problems when studying the role of metals in biology. As a complementary tool to optical and electron microscopes, developments and studies have demonstrated the unique capabilities of multi-keV microscopy: namely, an ultra-low detection limit, large penetration depth, chemical sensitivity and three-dimensional imaging capabilities. More recently, the capabilities have been extended towards sub-100nm lateral resolutions, thus enabling sub-cellular chemical imaging. Possibilities offered by these techniques in the biomedical field are described through examples of applications performed at the ESRF synchrotron-based microspectroscopy platform (ID21 and ID22 beamlines).
Automated tracing of microtubules in electron tomograms of plastic embedded samples of Caenorhabditis elegans embryos.
Venue: Journal of structural biology

The ability to rapidly assess microtubule number in 3D image stacks from electron tomograms is essential for collecting statistically meaningful data sets. Here we implement microtubule tracing using 3D template matching. We evaluate our results by comparing the automatically traced centerlines to manual tracings in a large number of electron tomograms of the centrosome of the early Caenorhabditis elegans embryo. Furthermore, we give a qualitative description of the tracing results for three other types of samples. For dual-axis tomograms, the automatic tracing yields 4% false negatives and 8% false positives on average. For single-axis tomograms, the accuracy of tracing is lower (16% false negatives and 14% false positives) due to the missing wedge in electron tomography. We also implemented an editor specifically designed for correcting the automatic tracing. Besides, this editor can be used for annotating microtubules. The automatic tracing together with a manual correction significantly reduces the amount of manual labor for tracing microtubule centerlines so that large-scale analysis of microtubule network properties becomes feasible.
Combined use of hard X-ray phase contrast imaging and X-ray fluorescence microscopy for sub-cellular metal quantification.
Venue: Journal of structural biology

Hard X-ray fluorescence microscopy and magnified phase contrast imaging are combined to obtain quantitative maps of the projected metal concentration in whole cells. The experiments were performed on freeze dried cells at the nano-imaging station ID22NI of the European Synchrotron Radiation Facility (ESRF). X-ray fluorescence analysis gives the areal mass of most major, minor and trace elements; it is validated using a biological standard of known composition. Quantitative phase contrast imaging provides maps of the projected mass and is validated using calibration samples and through comparison with Atomic Force Microscopy and Scanning Transmission Ion Microscopy. Up to now, absolute quantification at the sub-cellular level was impossible using X-ray fluorescence microscopy but can be reached with the use of the proposed approach.
Cryo X-ray nano-tomography of vaccinia virus infected cells
Venue: Journal of Structural Biology

Abstract
 
 We have performed full-field cryo X-ray microscopy in the water window photon energy range on vaccinia virus (VACV) infected cells to produce tomographic reconstructions. PtK2 cells were infected with a GFP-expressing VACV strain and frozen by plunge fast freezing. The infected cells were selected by light fluorescence microscopy of the GFP marker and subsequently imaged in the X-ray microscope under cryogenic conditions. Tomographic tilt series of X-ray images were used to yield three-dimensional reconstructions showing different cell organelles (nuclei, mitochondria, filaments), together with other structures derived from the virus infection. Among them, it was possible to detect viral factories and two types of viral particles related to different maturation steps of VACV (immature and mature particles), which were compared to images obtained by standard electron microscopy of the same type of cells. In addition, the effect of radiation damage during X-ray tomographic acquisition was analyzed. Thin sections studied by electron microscopy revealed that the morphological features of the cells do not present noticeable changes after irradiation. Our findings show that cryo X-ray nano-tomography is a powerful tool for collecting three-dimensional structural information from frozen, unfixed, unstained whole cells with sufficient resolution to detect different virus particles exhibiting distinct maturation levels.
D-CAT: Density and Clustering Annotation Tool for three dimensional electron microscopic volumes.
Venue: Journal of structural biology

Three dimensional (3D) electron microscopy techniques have become valuable tools for investigating cellular architecture and the processes that govern it. A vast amount of information is available in every 3D tomogram but the options for presenting this information in a clear and visually appealing way are limited. To address this, we developed D-CAT; a MatLab-application to accurately visualize the distribution of membrane proteins and/or membrane-bound structures. Presence (density) and distribution (clustering, depletion) are presented as color-coded areas on membranes. By using IMOD models both as input and output format, we ensure that the application fits within workflows common in the field of 3D electron microscopy.
Toll-like receptor 5 forms asymmetric dimers in the absence of flagellin.
Venue: Journal of structural biology

The structure of full-length human TLR5 determined by electron microscopy single-particle image reconstruction at 26Å resolution shows that TLR5 forms an asymmetric homodimer via ectodomain interactions. The structure shows that like TLR9, TLR5 dimerizes in the absence of ligand. The asymmetry of the dimer suggests that TLR5 may recognize two flagellin molecules cooperatively to establish an optimal flagellin response threshold. A TLR5 homology model was generated and fitted into the electron microscopy structure. All seven predicted N-linked glycosylation sites are exposed on the molecular surface, away from the dimer interface. Glycosylation at the first five sites was confirmed by tandem mass spectrometry. Two aspartate residues proposed to interact with flagellin (Asp294 and Asp366) are sterically occluded by a glycan at position 342. In contrast, the central region of the ectodomains near the dimer interface is unobstructed by glycans. Ligand binding in this region would be consistent with the ligand binding sites of other TLRs.
Molecular insights into substrate specificity and thermal stability of a bacterial GH5-CBM27 endo-1,4-β-D-mannanase.
Venue: Journal of structural biology

The breakdown of β-1,4-mannoside linkages in a variety of mannan-containing polysaccharides is of great importance in industrial processes such as kraft pulp delignification, food processing and production of second-generation biofuels, which puts a premium on studies regarding the prospection and engineering of β-mannanases. In this work, a two-domain β-mannanase from Thermotoga petrophila that encompasses a GH5 catalytic domain with a C-terminal CBM27 accessory domain, was functionally and structurally characterized. Kinetic and thermal denaturation experiments showed that the CBM27 domain provided thermo-protection to the catalytic domain, while no contribution on enzymatic activity was observed. The structure of the catalytic domain determined by SIRAS revealed a canonical (α/β)(8)-barrel scaffold surrounded by loops and short helices that form the catalytic interface. Several structurally related ligand molecules interacting with TpMan were solved at high-resolution and resulted in a wide-range representation of the subsites forming the active-site cleft with residues W134, E198, R200, E235, H283 and W284 directly involved in glucose binding.