nils-herrmann/clean_scirepeval huggingface dataset:
Cryo-tomography tilt-series alignment with consideration of the beam-induced sample motion
Venue: Journal of structural biology

Recent evidence suggests that the beam-induced motion of the sample during tilt-series acquisition is a major resolution-limiting factor in electron cryo-tomography (cryoET). It causes suboptimal tilt-series alignment and thus deterioration of the reconstruction quality. Here we present a novel approach to tilt-series alignment and tomographic reconstruction that considers the beam-induced sample motion through the tilt-series. It extends the standard fiducial-based alignment approach in cryoET by introducing quadratic polynomials to model the sample motion. The model can be used during reconstruction to yield a motion-compensated tomogram. We evaluated our method on various datasets with different sample sizes. The results demonstrate that our method could be a useful tool to improve the quality of tomograms and the resolution in cryoET.
Structural characterization of 14-3-3ζ in complex with the human Son of sevenless homolog 1 (SOS1).
Venue: Journal of structural biology

The deviant Ras activation machinery is found in approximately 30% of all human cancers. SOS1 is an important protagonist of this pathway that plays a key-role in aberrant cell proliferation and differentiation. Interaction of SOS1 with 14-3-3 proteins modulates SOS1 activity in Ras-MAPK signaling. In the present study, we analyze the 14-3-3/SOS1 protein-protein interaction (PPI) by different biochemical assays and report the high resolution crystal structure of a 13-mer motif of SOS1 bound to 14-3-3ζ. These structural and functional insights are important for the evaluation of this PPI interface for small-molecule stabilization as a new starting point for modulating the Ras-Raf-MAPK pathway.
Comparison of 3D cellular imaging techniques based on scanned electron probes: Serial block face SEM vs. Axial bright-field STEM tomography.
Venue: Journal of structural biology

Microscopies based on focused electron probes allow the cell biologist to image the 3D ultrastructure of eukaryotic cells and tissues extending over large volumes, thus providing new insight into the relationship between cellular architecture and function of organelles. Here we compare two such techniques: electron tomography in conjunction with axial bright-field scanning transmission electron microscopy (BF-STEM), and serial block face scanning electron microscopy (SBF-SEM). The advantages and limitations of each technique are illustrated by their application to determining the 3D ultrastructure of human blood platelets, by considering specimen geometry, specimen preparation, beam damage and image processing methods. Many features of the complex membranes composing the platelet organelles can be determined from both approaches, although STEM tomography offers a higher ∼3 nm isotropic pixel size, compared with ∼5 nm for SBF-SEM in the plane of the block face and ∼30 nm in the perpendicular direction. In this regard, we demonstrate that STEM tomography is advantageous for visualizing the platelet canalicular system, which consists of an interconnected network of narrow (∼50-100 nm) membranous cisternae. In contrast, SBF-SEM enables visualization of complete platelets, each of which extends ∼2 µm in minimum dimension, whereas BF-STEM tomography can typically only visualize approximately half of the platelet volume due to a rapid non-linear loss of signal in specimens of thickness greater than ∼1.5 µm. We also show that the limitations of each approach can be ameliorated by combining 3D and 2D measurements using a stereological approach.
Crystal structure of thebaine 6-O-demethylase from the morphine biosynthesis pathway.
Venue: Journal of structural biology

Thebaine 6-O-demethylase (T6ODM) from Papaver somniferum (opium poppy), which belongs to the non-heme 2-oxoglutarate/Fe(II)-dependent dioxygenases (ODD) family, is a key enzyme in the morphine biosynthesis pathway. Initially, T6ODM was characterized as an enzyme catalyzing O-demethylation of thebaine to neopinone and oripavine to morphinone. However, the substrate range of T6ODM was recently expanded to a number of various benzylisoquinoline alkaloids. Here, we present crystal structures of T6ODM in complexes with 2-oxoglutarate (T6ODM:2OG, PDB: 5O9W) and succinate (T6ODM:SIN, PDB: 5O7Y). Both metal and 2OG binding sites display similarity to other proteins from the ODD family, but T6ODM is characterized by an exceptionally large substrate binding cavity, whose volume can partially explain the promiscuity of this enzyme. Moreover, the size of the cavity allows for binding of multiple molecules at once, posing a question about the substrate-driven specificity of the enzyme.
Cep120 promotes microtubule formation through a unique tubulin binding C2 domain.
Venue: Journal of structural biology

Centrioles are microtubule-based structures that play essential roles in cell division and cilia biogenesis. Cep120 is an important protein for correct centriole formation and mutations in the Cep120 gene cause severe human diseases like Joubert syndrome and complex ciliopathies. Here, we show that Cep120 contains three consecutive C2 domains that are followed by a coiled-coil dimerization domain. Surprisingly, unlike the classical C2 domains, all three Cep120 C2 domains lack calcium- and phospholipid-binding activities. However, biophysical and biochemical assays revealed that the N-terminal Cep120 C2 domain (C2A) binds to both tubulin and microtubules, and promotes microtubule formation. Structural analyses coupled with mutagenesis identified a highly conserved, positively charged residue patch on the surface of Cep120 C2A, which mediates the interaction with tubulin and microtubules. Together, our results establish Cep120 C2A as a unique microtubule-binding domain. They further provide insights into the molecular mechanism of Cep120 during centriole biogenesis.
Spotiton: New features and applications.
Venue: Journal of structural biology

We present an update describing new features and applications of Spotiton, a novel instrument for vitrifying samples for cryoEM. We have used Spotiton to prepare several test specimens that can be reconstructed using routine single particle analysis to ∼3 Å resolution, indicating that the process has no apparent deleterious effect on the sample integrity. The system is now in routine and continuous use in our lab and has been used to successfully vitrify a wide variety of samples.
Volta phase plate data collection facilitates image processing and cryo-EM structure determination.
Venue: Journal of structural biology

A current bottleneck in structure determination of macromolecular complexes by cryo electron microscopy (cryo-EM) is the large amount of data needed to obtain high-resolution 3D reconstructions, including through sorting into different conformations and compositions with advanced image processing. Additionally, it may be difficult to visualize small ligands that bind in sub-stoichiometric levels. Volta phase plates (VPP) introduce a phase shift in the contrast transfer and drastically increase the contrast of the recorded low-dose cryo-EM images while preserving high frequency information. Here we present a comparative study to address the behavior of different data sets during image processing and quantify important parameters during structure refinement. The automated data collection was done from the same human ribosome sample either as a conventional defocus range dataset or with a Volta phase plate close to focus (cfVPP) or with a small defocus (dfVPP). The analysis of image processing parameters shows that dfVPP data behave more robustly during cryo-EM structure refinement because particle alignments, Euler angle assignments and 2D & 3D classifications behave more stably and converge faster. In particular, less particle images are required to reach the same resolution in the 3D reconstructions. Finally, we find that defocus range data collection is also applicable to VPP. This study shows that data processing and cryo-EM map interpretation, including atomic model refinement, are facilitated significantly by performing VPP cryo-EM, which will have an important impact on structural biology.
Structure of the Bacillus anthracis dTDP-l-rhamnose biosynthetic pathway enzyme: dTDP-α-d-glucose 4,6-dehydratase, RfbB.
Venue: Journal of structural biology

Many bacteria require l-rhamnose as a key cell wall component. This sugar is transferred to the cell wall using an activated donor dTDP-l-rhamnose, which is produced by the dTDP-l-rhamnose biosynthetic pathway. We determined the crystal structure of the second enzyme of this pathway dTDP-α-d-glucose 4,6-dehydratase (RfbB) from Bacillus anthracis. Interestingly, RfbB only crystallized in the presence of the third enzyme of the pathway RfbC; however, RfbC was not present in the crystal. Our work represents the first complete structural characterization of the four proteins of this pathway in a single Gram-positive bacterium.
Allosteric effects in bacteriophage HK97 procapsids revealed directly from covariance analysis of cryo EM data.
Venue: Journal of structural biology

The information content of cryo EM data sets exceeds that of the electron scattering potential (cryo EM) density initially derived for structure determination. Previously we demonstrated the power of data variance analysis for characterizing regions of cryo EM density that displayed functionally important variance anomalies associated with maturation cleavage events in Nudaurelia Omega Capensis Virus and the presence or absence of a maturation protease in bacteriophage HK97 procapsids. Here we extend the analysis in two ways. First, instead of imposing icosahedral symmetry on every particle in the data set during the variance analysis, we only assume that the data set as a whole has icosahedral symmetry. This change removes artifacts of high variance along icosahedral symmetry axes, but retains all of the features previously reported in the HK97 data set. Second we present a covariance analysis that reveals correlations in structural dynamics (variance) between the interior of the HK97 procapsid with the protease and regions of the exterior (not seen in the absence of the protease). The latter analysis corresponds well with hydrogen deuterium exchange studies previously published that reveal the same correlation.
Impact of intracellular ionic strength on dimer binding in the NF-kB Inducing kinase.
Venue: Journal of structural biology

Improper signaling of the nuclear factor-κB (NF-κB) pathway plays a critical role in many inflammatory disease states including cancer, stroke, and viral infections. Although the signaling pathways are known, how these molecular mechanisms respond to changes in the intracellular microenvironment such as pH, ionic strength, and temperature, remains elusive. Molecular dynamics simulations were employed to differentiate the structural dynamics of the NF-κB Inducing Kinase (NIK), a protein kinase responsible for invoking the non-canonical NF-κB pathway, in its native and mutant form, and in the absence and presence of salt concentration in efforts to probe whether changes in the ionic environment stabilize or destabilize the NIK dimer. Analyses of structure-activity and conformational-activity relationships indicate that the protein-protein interactions are sensitive to changes in the ionic strength. Ligand binding pockets as well as regions between the oligomer interface either compress or expand, affecting both local and distal intermolecular interactions that result in stabilization or destabilization in the protein assembly.
Optimizing "self-wicking" nanowire grids.
Venue: Journal of structural biology

We have developed a self-blotting TEM grid for use with a novel instrument for vitrifying samples for cryo-electron microscopy (cryoEM). Nanowires are grown on the copper surface of the grid using a simple chemical reaction and the opposite smooth side is used to adhere to a holey sample substrate support, for example carbon or gold. When small volumes of sample are applied to the nanowire grids the wires effectively act as blotting paper to rapidly wick away the liquid, leaving behind a thin film. In this technical note, we present a detailed description of how we make these grids using a variety of substrates fenestrated with either lacey or regularly spaced holes. We explain how we characterize the quality of the grids and we describe their behavior under a variety of conditions.
Comparative structural and enzymatic studies on Salmonella typhimurium diaminopropionate ammonia lyase reveal its unique features.
Venue: Journal of structural biology

Cellular metabolism of amino acids is controlled by a large number of pyridoxal 5'-phosphate (PLP) dependent enzymes. Diaminopropionate ammonia lyase (DAPAL), a fold type II PLP-dependent enzyme, degrades both the D and L forms of diaminopropionic acid (DAP) to pyruvate and ammonia. Earlier studies on the Escherichia coli DAPAL (EcDAPAL) had suggested that a disulfide bond located close to the active site may be crucial for maintaining the geometry of the substrate entry channel and the active site. In order to obtain further insights into the catalytic properties of DAPAL, structural and functional studies on Salmonella typhimurium DAPAL (StDAPAL) were initiated. The three-dimensional X-ray crystal structure of StDAPAL was determined at 2.5 Å resolution. As expected, the polypeptide fold and dimeric organization of StDAPAL is similar to those of EcDAPAL. A phosphate group was located in the active site of StDAPAL and expulsion of this phosphate is probably essential to bring Asp125 to a conformation suitable for proton abstraction from the substrate (D-DAP). The unique disulfide bond of EcDAPAL was absent in StDAPAL, although the enzyme displayed comparable catalytic activity. Site directed mutagenesis of the cysteine residues involved in disulfide bond formation in EcDAPAL followed by functional and biophysical studies further confirmed that the disulfide bond is not necessary either for substrate binding or for catalysis. The activity of StDAPAL but not EcDAPAL was enhanced by monovalent cations suggesting subtle differences in the active site geometries of these two closely related enzymes.
A novel small molecule displays two different binding modes during inhibiting H1N1 influenza A virus neuraminidases.
Venue: Journal of structural biology

Neuraminidase (NA) inhibitors can suppress NA activity to block the release of progeny virions and are effective against influenza viruses. As potential anti-flu drugs with unique functions, NA inhibitors are greatly concerned by the worldwide scientists. It has been reported recently that one of the novel quindoline derivatives named 7a, could inhibit both A/Puerto Rico/8/34 (H1N1) NA (NAPR) and A/California/04/09 (H1N1) NA (NACA). However, potential structure differences in the active site could be easily detected between the NAPR and NACA according to the flexibilities of their 150-loops located catalytic site. And no obvious 150-cavity could be observed in NACA crystal structure. In order to explore whether 7a could trigger the inhibition against these two NAs in the same way, a serial molecular dynamics simulation approach were applied in this study. The results indicated that 7a could be adopted under a relatively extended pose in the active center of NAPR. While in NACA-7a complex, the derivate preferred to be recognized and located on the side of active center. Interestingly, the potential of 7a was also found to be able to change the flexibility of the 150-loop in NACA that is absent of 150-cavity. Furthermore, a 150-cavity-like architecture could be induced in the active site of NACA. The results of this study revealed two kinds of binding modes of this novel small molecule inhibitor against NAs that might provide a theoretical basis for proposing novel inhibition mechanism and developing future influenza A virus inhibitors.
A convolutional autoencoder approach for mining features in cellular electron cryo-tomograms and weakly supervised coarse segmentation
Venue: Journal of structural biology

Cellular electron cryo-tomography enables the 3D visualization of cellular organization in the near-native state and at submolecular resolution. However, the contents of cellular tomograms are often complex, making it difficult to automatically isolate different in situ cellular components. In this paper, we propose a convolutional autoencoder-based unsupervised approach to provide a coarse grouping of 3D small subvolumes extracted from tomograms. We demonstrate that the autoencoder can be used for efficient and coarse characterization of features of macromolecular complexes and surfaces, such as membranes. In addition, the autoencoder can be used to detect non-cellular features related to sample preparation and data collection, such as carbon edges from the grid and tomogram boundaries. The autoencoder is also able to detect patterns that may indicate spatial interactions between cellular components. Furthermore, we demonstrate that our autoencoder can be used for weakly supervised semantic segmentation of cellular components, requiring a very small amount of manual annotation.
Structural characterization of SpoIIIAB sporulation-essential protein in Bacillus subtilis.
Venue: Journal of structural biology

Endospore formation in the Gram-positive bacterium Bacillus subtilis initiates in response to nutrient depletion and involves a series of morphological changes that result in the creation of a dormant spore. Early in this developmental process, the cell undergoes an asymmetric cell division that produces the larger mother cell and smaller forespore, the latter destined to become the mature spore. The mother cell septal membrane then engulfs the forespore, at which time an essential channel, the so-called feeding-tube apparatus, is thought to cross both membranes to create a direct conduit between the cells. At least nine proteins are required to form this channel including SpoIIQ under forespore control and SpoIIIAA-AH under the mother cell control. Several of these proteins share similarity to components of Type-II, -III and -IV secretion systems as well as the flagellum from Gram-negative bacteria. Here we report the X-ray crystallographic structure of the cytosolic domain of SpoIIIAB to 2.3 Å resolution. This domain adopts a conserved, secretion-system related fold of a six membered anti-parallel helical bundle with a positively charged membrane-interaction face at one end and a small groove at the other end that may serve as a binding site for partner proteins in the assembled apparatus. We analyzed and identified potential interaction interfaces by structure-guided mutagenesis in vivo. Furthermore, we were able to identify a remarkable structural homology to the C-subunit of a bacterial V-ATPase. Collectively, our data provides new insight into the possible roles of SpoIIIAB protein within the secretion-like apparatus essential to bacterial sporulation.
High resolution crystal structures of Clostridium botulinum neurotoxin A3 and A4 binding domains.
Venue: Journal of structural biology

Clostridium botulinum neurotoxins (BoNTs) cause the life-threatening condition, botulism. However, while they have the potential to cause serious harm, they are increasingly being utilised for therapeutic applications. BoNTs comprise of seven distinct serotypes termed BoNT/A through BoNT/G, with the most widely characterised being sub-serotype BoNT/A1. Each BoNT consists of three structurally distinct domains, a binding domain (HC), a translocation domain (HN), and a proteolytic domain (LC). The HC domain is responsible for the highly specific targeting of the neurotoxin to neuronal cell membranes. Here, we present two high-resolution structures of the binding domain of subtype BoNT/A3 (HC/A3) and BoNT/A4 (HC/A4) at 1.6 Å and 1.34 Å resolution, respectively. The structures of both proteins share a high degree of similarity to other known BoNT HC domains whilst containing some subtle differences, and are of benefit to research into therapeutic neurotoxins with novel characteristics.
Translocation of Epidermal Growth Factor (EGF) to the nucleus has distinct kinetics between adipose tissue-derived mesenchymal stem cells and a mesenchymal cancer cell lineage.
Venue: Journal of structural biology

Nuclear Epidermal Growth Factor Receptor (EGFR) has been associated with worse prognosis and treatment resistance for several cancer types. After Epidermal Growth Factor (EGF) binding, the ligand-receptor complex can translocate to the nucleus where it functions in oncological processes. By three-dimensional quantification analysis of super-resolution microscopy images, we verified the translocation kinetics of fluorescent conjugated EGF to the nucleus in two mesenchymal cell types: human adipose tissue-derived stem cells (hASC) and SK-HEP-1 tumor cells. The number of EGF clusters in the nucleus does not change after 10 min of stimulation with EGF in both cells. The total volume occupied by EGF clusters in the nucleus of hASC also does not change after 10 min of stimulation with EGF. However, the total volume of EGF clusters increases only after 20 min in SK-HEP-1 cells nuclei. In these cells the nuclear volume occupied by EGF is 3.2 times higher than in hASC after 20 min of stimulation, indicating that translocation kinetics of EGF differs between these two cell types. After stimulation, EGF clusters assemble in larger clusters in the cell nucleus in both cell types, which suggests specific sub-nuclear localizations of the receptor. Super-resolution microscopy images show that EGF clusters are widespread in the nucleoplasm, and can be localized in nuclear envelope invaginations, and in the nucleoli. The quantitative study of EGF-EGFR complex translocation to the nucleus may help to unravel its roles in health and pathological conditions, such as cancer.
Crystal structure of cystathionine β-synthase from honeybee Apis mellifera.
Venue: Journal of structural biology

Cystathionine β-synthase (CBS), the key enzyme in the transsulfuration pathway, links methionine metabolism to the biosynthesis of cellular redox controlling molecules. CBS catalyzes the pyridoxal-5'-phosphate-dependent condensation of serine and homocysteine to form cystathionine, which is subsequently converted into cysteine. Besides maintaining cellular sulfur amino acid homeostasis, CBS also catalyzes multiple hydrogen sulfide-generating reactions using cysteine and homocysteine as substrates. In mammals, CBS is activated by S-adenosylmethionine (AdoMet), where it can adopt two different conformations (basal and activated), but exists as a unique highly active species in fruit fly Drosophila melanogaster. Here we present the crystal structure of CBS from honeybey Apis mellifera, which shows a constitutively active dimeric species and let explain why the enzyme is not allosterically regulated by AdoMet. In addition, comparison of available CBS structures unveils a substrate-induced closure of the catalytic cavity, which in humans is affected by the AdoMet-dependent regulation and likely impaired by the homocystinuria causing mutation T191M.