nils-herrmann/clean_scirepeval huggingface dataset:
Improving the visualization of cryo-EM density reconstructions.
Venue: Journal of structural biology

Cryo-electron microscopy yields 3D density maps of macromolecules from single-particle images, tomograms, or 2D crystals. An optimal visualization of the density map is important for its proper interpretation. We have developed a method to improve the visualization of density maps by using general statistical information about proteins for the sharpening process. In particular, the packing density of atoms is highly similar between different proteins, which allows for building a pseudo-atomic model to approximate the true mass distribution. From this model the radial structure factor and density value histogram are estimated and applied as constraints to the 3D reconstruction in reciprocal- and real-space, respectively. Interestingly, similar improvements are obtained when using the correct radial structure factor and density value histogram from a crystal structure. Thus, the estimated pseudo-atomic model yields a sufficiently accurate mass distribution to optimally sharpen a density map.
Molecular mechanism for the action of the anti-CD44 monoclonal antibody MEM-85.
Venue: Journal of structural biology

The hyaluronate receptor CD44 plays role in cell adhesion and migration and is involved in tumor metastasis. The extracellular domain of CD44 comprises the hyaluronate-binding domain (HABD) and the membrane-proximal stem region; the short intracellular portion interacts with adaptor proteins and triggers signaling pathways. Binding of hyaluronate to CD44 HABD induces an allosteric conformational change, which results in CD44 shedding. A poorly characterized epitope in human CD44 HABD is recognized by the murine monoclonal antibody MEM-85, which cross-blocks hyaluronate binding to CD44 and also induces CD44 shedding. MEM-85 is of therapeutic interest, as it inhibits growth of lung cancer cells in murine models. In this work, we employed a combination of biophysical methods to determine the MEM-85 binding epitope in CD44 HABD and to provide detailed insight into the mechanism of MEM-85 action. In particular, we constructed a single-chain variable fragment (scFv) of MEM-85 as a tool for detailed characterization of the CD44 HABD-antibody complex and identified residues within CD44 HABD involved in the interaction with scFv MEM-85 by NMR spectroscopy and mutational analysis. In addition, we built a rigid body model of the CD44 HABD-scFv MEM-85 complex using a low-resolution structure obtained by small-angle X-ray scattering. The MEM-85 epitope is situated in the C-terminal part of CD44 HABD, rather than the hyaluronate-binding groove, and the binding of MEM-85 induces a structural reorganization similar to that induced by hyaluronate. Therefore, the mechanism of MEM-85 cross-blocking of hyaluronate binding is likely of an allosteric, relay-like nature.
Label-free microscopy and stress responses reveal the functional organization of Pseudodiaptomus marinus copepod myofibrils.
Venue: Journal of structural biology

Pseudodiaptomus marinus copepods are small crustaceans living in estuarine areas endowed with exceptional swimming and adaptative performances. Since the external cuticle acts as an impermeable barrier for most dyes and molecular tools for labeling copepod proteins with fluorescent tags are not available, imaging cellular organelles in these organisms requires label free microscopy. Complementary nonlinear microscopy techniques have been used to investigate the structure and the response of their myofibrils to abrupt changes of temperature or/and salinity. In contrast with previous observations in vertebrates and invertebrates, the flavin autofluorescence which is a signature of mitochondria activity and the Coherent Anti-Stokes Raman Scattering (CARS) pattern assigned to T-tubules overlapped along myofibrils with the second harmonic generation (SHG) striated pattern generated by myosin tails in sarcomeric A bands. Temperature jumps from 18 to 4 °C or salinity jumps from 30 to 15 psu mostly affected flavin autofluorescence. Severe salinity jumps from 30 to 0 psu dismantled myofibril organization with major changes both in the SHG and CARS patterns. After a double stress (from 18 °C/30 psu to 4° C/0 psu) condensed and distended regions appeared within single myofibrils, with flavin autofluorescence bands located between sarcomeric A bands. These results shed light on the interactions between the different functional compartments which provide fast acting excitation-contraction coupling and adequate power supply in copepods muscles.
Structure of EspB, a secreted substrate of the ESX-1 secretion system of Mycobacterium tuberculosis.
Venue: Journal of structural biology

Mycobacterium tuberculosis secretes multiple virulence factors during infection via the general Sec and Tat pathways, and via specialized ESX secretion systems, also referred to as type VII secretion systems. The ESX-1 secretion system is an important virulence determinant because deletion of ESX-1 leads to attenuation of M. tuberculosis. ESX-1 secreted protein B (EspB) contains putative PE (Pro-Glu) and PPE (Pro-Pro-Glu) domains, and a C-terminal domain, which is processed by MycP1 protease during secretion. We determined the crystal structure of PE-PPE domains of EspB, which represents an all-helical, elongated molecule closely resembling the structure of the PE25-PPE41 heterodimer despite limited sequence similarity. Also, we determined the structure of full-length EspB, which does not have interpretable electron density for the C-terminal domain confirming that it is largely disordered. Comparative analysis of EspB in cell lysate and culture filtrates of M. tuberculosis revealed that mature secreted EspB forms oligomers. Electron microscopy analysis showed that the N-terminal fragment of EspB forms donut-shaped particles. These data provide a rationale for the future investigation of EspB's role in M. tuberculosis pathogenesis.
Time-lapse imaging of morphological changes in a single neuron during the early stages of apoptosis using scanning ion conductance microscopy.
Venue: Journal of structural biology

Apoptosis plays an important role in many physiologic and pathologic conditions. The biochemical and morphological characteristics of apoptosis including cellular volume decrease, cell membrane blebbing, and phosphatidylserine translocation from the inner to the outer leaflet of the cell membrane are considered important events for phagocyte detection. Despite its importance, the relationship between the biological and morphological changes in a living cell has remained controversial. Scanning ion conductance microscopy is a suitable technique for investigating a series of these changes, because it allows us to observe the morphology of living cells without any mechanical interactions between the probe and the sample surface with a high resolution. Here, we investigated the biochemical and morphological changes in single neurons during the early stages of apoptosis, including apoptotic volume decrease, membrane blebbing and phosphatidylserine translocation, by using scanning ion conductance microscopy. Time-course imaging of apoptotic neurons showed there was a reduction in apoptotic volume after exposure to staurosporine and subsequent membrane bleb formation, which has a similar onset time to phosphatidylserine translocation. Our results show that a reduction in cellular volume is one of the earliest morphological changes in apoptosis, and membrane blebbing and phosphatidylserine translocation occur as subsequent biological and morphological changes. This is the first report to describe this series of morphological and biochemical changes ranging from an apoptotic volume decrease to membrane blebbing and PS translocation by scanning ion conductance microscopy (SICM). This new and direct imaging technique will provide new insight into the relationship between biochemical events inside a cell and cellular morphological changes.
Tissue-specific sequence and structural environments of lysine acetylation sites.
Venue: Journal of structural biology

Lysine acetylation is a widespread reversible post-translational modification that regulates a broad spectrum of biological activities across various cellular compartments, cell types, tissues, and disease states. While compartment-specific trends in lysine acetylation have recently been investigated, its tissue-specific preferences remain unexplored. Here we present a comprehensive tissue-based analysis of sequence and structural features of lysine acetylation sites (LASs) based on the recent experimental data of Lundby et al. (2012). We show that acetylated substrates are characterized by tissue-specific motifs both in linear amino acid sequence and in spatial environments. We further demonstrate that the general tendency of LASs to reside in ordered regions and, specifically, in α-helices, is also subject to tissue specific variation. In line with previous findings we show that LASs are generally more evolutionarily conserved than non-LASs, especially in proteins with known function and in structurally regular regions. On the other hand, as revealed by metabolic pathway analysis, LASs have diverse cellular functions in different tissues and are frequently associated with tissue-specific protein domains. These findings may imply the existence of tissue-specific lysine acetyltransferases (KATs) and lysine deacetylases (KDACs).
Directly reconstructing principal components of heterogeneous particles from cryo-EM images.
Venue: Journal of structural biology

Structural heterogeneity of particles can be investigated by their three-dimensional principal components. This paper addresses the question of whether, and with what algorithm, the three-dimensional principal components can be directly recovered from cryo-EM images. The first part of the paper extends the Fourier slice theorem to covariance functions showing that the three-dimensional covariance, and hence the principal components, of a heterogeneous particle can indeed be recovered from two-dimensional cryo-EM images. The second part of the paper proposes a practical algorithm for reconstructing the principal components directly from cryo-EM images without the intermediate step of calculating covariances. This algorithm is based on maximizing the posterior likelihood using the Expectation-Maximization algorithm. The last part of the paper applies this algorithm to simulated data and to two real cryo-EM data sets: a data set of the 70S ribosome with and without Elongation Factor-G (EF-G), and a data set of the influenza virus RNA dependent RNA Polymerase (RdRP). The first principal component of the 70S ribosome data set reveals the expected conformational changes of the ribosome as the EF-G binds and unbinds. The first principal component of the RdRP data set reveals a conformational change in the two dimers of the RdRP.
High throughput screening using acoustic droplet ejection to combine protein crystals and chemical libraries on crystallization plates at high density.
Venue: Journal of structural biology

We describe a high throughput method for screening up to 1728 distinct chemicals with protein crystals on a single microplate. Acoustic droplet ejection (ADE) was used to co-position 2.5nL of protein, precipitant, and chemicals on a MiTeGen in situ-1 crystallization plate™ for screening by co-crystallization or soaking. ADE-transferred droplets follow a precise trajectory which allows all components to be transferred through small apertures in the microplate lid. The apertures were large enough for 2.5nL droplets to pass through them, but small enough so that they did not disrupt the internal environment created by the mother liquor. Using this system, thermolysin and trypsin crystals were efficiently screened for binding to a heavy-metal mini-library. Fluorescence and X-ray diffraction were used to confirm that each chemical in the heavy-metal library was correctly paired with the intended protein crystal. A fragment mini-library was screened to observe two known lysozyme ligands using both co-crystallization and soaking. A similar approach was used to identify multiple, novel thaumatin binding sites for ascorbic acid. This technology pushes towards a faster, automated, and more flexible strategy for high throughput screening of chemical libraries (such as fragment libraries) using as little as 2.5nL of each component.
Osteocyte lacunar properties in rat cortical bone: Differences between lamellar and central bone.
Venue: Journal of structural biology

Recently, the roles of osteocytes in bone maintenance have gained increasing attention. Osteocytes reside in lacunae that are interconnected by canaliculi resulting in a vast cellular network within the mineralized bone matrix. As the structure of the lacuno-canalicular network is highly connected to osteocyte function, osteocyte lacunar properties such as volume, shape, orientation, and density are now frequently reported in studies investigating osteocyte activity. Despite this increasing interest in lacunar morphometrics, many studies show a large spread in such values, suggesting a large inter-species but also inter-site variation in lacunar properties. Here, osteocyte lacunae in rat cortical bone have been studied using synchrotron radiation micro computed tomography (SR μCT) and backscattered electron (BE) microscopy. Quantitative lacunar geometric characteristics are reported based on the synchrotron radiation data, differentiating between circumferential lamellar bone and a central, more disordered bone type. From these studies, no significant differences were found in lacunar volumes between lamellar and central bone, whereas significant differences in lacunar orientation, shape and density values were observed. The 3D nature of the SR μCT data sets furthermore revealed that lacunae in central bone, which appear to be poorly aligned in transverse 2D cross sections, are in fact highly aligned along the bone long axis. These results demonstrate the importance of using 3D methods to investigate anisotropic biological materials such as bone and that the appropriate choice of subregions for high resolution imaging is not trivial.
Large conformational fluctuations of the multi-domain xylanase Z of Clostridium thermocellum.
Venue: Journal of structural biology

The cellulosome is a multi-enzyme machinery which efficiently degrades plant cell-wall polysaccharides. The multiple domains of the cellulosome complexes are often tethered to one another by intrinsically disordered regions. The properties and functions of these disordered linkers are unknown to a large extent. In this work, we study the conformational variability of one component of the cellulosome - the multi-domain xylanase Z (XynZ) of Clostridium thermocellum. We use a coarse-grained protein model to efficiently simulate conformations of the enzyme. Our simulation results are in excellent agreement with data from small angle X-ray scattering experiments, which validates the simulation outcome. Both in the presence and absence of the cohesin domain, the XynZ enzyme appears to be flexible in the sense that it takes various compact and extended conformations. The physical interactions between the individual domains are rather weak and transient, and the XynZ enzyme is held together mainly by the flexible linkers connecting the domains. The end-to-end distance distributions for the flexible linkers can be rationalized by the excluded volume effect. Taken together, our results provide a detailed picture of the conformational ensemble of the XynZ enzyme in solution.
New views of the Toxoplasma gondii parasitophorous vacuole as revealed by Helium Ion Microscopy (HIM).
Venue: Journal of structural biology

The Helium Ion Microscope (HIM) is a new technology that uses a highly focused helium ion beam to scan and interact with the sample, which is not coated. The images have resolution and depth of field superior to field emission scanning electron microscopes. In this paper, we used HIM to study LLC-MK2 cells infected with Toxoplasma gondii. These samples were chemically fixed and, after critical point drying, were scraped with adhesive tape to expose the inner structure of the cell and parasitophorous vacuoles. We confirmed some of the previous findings made by field emission-scanning electron microscopy and showed that the surface of the parasite is rich in structures suggestive of secretion, that the nanotubules of the intravacuolar network (IVN) are not always straight, and that bifurcations are less frequent than previously thought. Fusion of the tubules with the parasite membrane or the parasitophorous vacuole membrane (PVM) was also infrequent. Tiny adhesive links were observed for the first time connecting the IVN tubules. The PVM showed openings of various sizes that even allowed the observation of endoplasmic reticulum membranes in the cytoplasm of the host cell. These findings are discussed in relation to current knowledge on the cell biology of T. gondii.
Structural insights on cholesterol endosynthesis: Binding of squalene and 2,3-oxidosqualene to supernatant protein factor.
Venue: Journal of structural biology

We present the crystal structures of the SEC14-like domain of supernatant protein factor (SPF) in complex with squalene and 2,3-oxidosqualene. The structures were resolved at 1.75Å (complex with squalene) and 1.6Å resolution (complex with 2,3-oxidosqualene), leading in both cases to clear images of the protein/substrate interactions. Ligand binding is facilitated by removal of the Golgi-dynamics (GOLD) C-terminal domain of SPF, which, as shown in previous structures of the apo-protein, blocked the opening of the binding pocket to the exterior. Both substrates bind into a large hydrophobic cavity, typical of such lipid-transporter family. Our structures report no specific recognition mode for the epoxide group. In fact, for both molecules, ligand affinity is dominated by hydrophobic interactions, and independent investigations by computational models or differential scanning micro-calorimetry reveal similar binding affinities for both ligands. Our findings elucidate the molecular bases of the role of SPF in sterol endo-synthesis, supporting the original hypothesis that SPF is a facilitator of substrate flow within the sterol synthetic pathway. Moreover, our results suggest that the GOLD domain acts as a regulator, as its conformational displacement must occur to favor ligand binding and release during the different synthetic steps.
Local amino acid sequence patterns dominate the heterogeneous phenotype for the collagen connective tissue disease Osteogenesis Imperfecta resulting from Gly mutations.
Venue: Journal of structural biology

Osteogenesis Imperfecta (OI), a hereditary connective tissue disease in collagen that arises from a single Gly → X mutation in the collagen chain, varies widely in phenotype from perinatal lethal to mild. It is unclear why there is such a large variation in the severity of the disease considering the repeating (Gly-X-Y)n sequence and the uniform rod-like structure of collagen. We systematically evaluate the effect of local (Gly-X-Y)n sequence around the mutation site on OI phenotype using integrated bio-statistical approaches, including odds ratio analysis and decision tree modeling. We show that different Gly → X mutations have different local sequence patterns that are correlated with lethal and nonlethal phenotypes providing a mechanism for understanding the sensitivity of local context in defining lethal and non-lethal OI. A number of important trends about which factors are related to OI phenotypes are revealed by the bio-statistical analyses; most striking is the complementary relationship between the placement of Pro residues and small residues and their correlation to OI phenotype. When Pro is present or small flexible residues are absent nearby a mutation site, the OI case tends to be lethal; when Pro is present or small flexible residues are absent further away from the mutation site, the OI case tends to be nonlethal. The analysis also reveals the dominant role of local sequence around mutation sites in the Major Ligand Binding Regions that are primarily responsible for collagen binding to its receptors and shows that non-lethal mutations are highly predicted by local sequence considerations alone whereas lethal mutations are not as easily predicted and may be a result of more complex interactions. Understanding the sequence determinants of OI mutations will enhance genetic counseling and help establish which steps in the collagen hierarchy to target for drug therapy.
Helium ion microscopy and ultra-high-resolution scanning electron microscopy analysis of membrane-extracted cells reveals novel characteristics of the cytoskeleton of Giardia intestinalis.
Venue: Journal of structural biology

Giardia intestinalis presents a complex microtubular cytoskeleton formed by specialized structures, such as the adhesive disk, four pairs of flagella, the funis and the median body. The ultrastructural organization of the Giardia cytoskeleton has been analyzed using different microscopic techniques, including high-resolution scanning electron microscopy. Recent advances in scanning microscopy technology have opened a new venue for the characterization of cellular structures and include scanning probe microscopy techniques such as ultra-high-resolution scanning electron microscopy (UHRSEM) and helium ion microscopy (HIM). Here, we studied the organization of the cytoskeleton of G. intestinalis trophozoites using UHRSEM and HIM in membrane-extracted cells. The results revealed a number of new cytoskeletal elements associated with the lateral crest and the dorsal surface of the parasite. The fine structure of the banded collar was also observed. The marginal plates were seen linked to a network of filaments, which were continuous with filaments parallel to the main cell axis. Cytoplasmic filaments that supported the internal structures were seen by the first time. Using anti-actin antibody, we observed a labeling in these filamentous structures. Taken together, these data revealed new surface characteristics of the cytoskeleton of G. intestinalis and may contribute to an improved understanding of the structural organization of trophozoites.
Single particle tomography in EMAN2.
Venue: Journal of structural biology

Single particle tomography (SPT or subtomogram averaging) offers a powerful alternative to traditional 2-D single particle reconstruction for studying conformationally or compositionally heterogeneous macromolecules. It can also provide direct observation (without labeling or staining) of complexes inside cells at nanometer resolution. The development of computational methods and tools for SPT remains an area of active research. Here we present the EMAN2.1 SPT toolbox, which offers a full SPT processing pipeline, from particle picking to post-alignment analysis of subtomogram averages, automating most steps. Different algorithm combinations can be applied at each step, providing versatility and allowing for procedural cross-testing and specimen-specific strategies. Alignment methods include all-vs-all, binary tree, iterative single-model refinement, multiple-model refinement, and self-symmetry alignment. An efficient angular search, Graphic Processing Unit (GPU) acceleration and both threaded and distributed parallelism are provided to speed up processing. Finally, automated simulations, per particle reconstruction of subtiltseries, and per-particle Contrast Transfer Function (CTF) correction have been implemented. Processing examples using both real and simulated data are shown for several structures.