nils-herrmann/clean_scirepeval huggingface dataset:
Structural insight into the mechanism of action of antimicrobial peptide BMAP-28(1-18) and its analogue mutBMAP18.
Venue: Journal of structural biology

Structural characterization of BMAP-28(1-18), a potent bovine myeloid antimicrobial peptide can aid in understanding its mechanism of action at molecular level. We report NMR structure of the BMAP-28(1-18) and its mutated analogue mutBMAP18 in SDS micelles. Structural comparison of the peptides bound to SDS micelles and POPE-POPG vesicles using circular dichroism, suggest that structures in the two lipid preparations are similar. Antimicrobial assays show that even though both these peptides adopt helical conformation, BMAP-28(1-18) is more potent than mutBMAP18 in killing bacterial cells. Our EM images clearly indicate that the peptides target the bacterial cell membrane resulting in leakage of its contents. The structural basis for difference in activity between these peptides was investigated by molecular dynamics simulations. Inability of the mutBMAP18 to retain its helical structure in presence of POPE:POPG membrane as opposed to the BMAP-28(1-18) at identical peptide/lipid ratios could be responsible for its decreased activity. Residues Ser5, Arg8 and Arg12 of the BMAP-28(1-18) are crucial for its initial anchoring to the bilayer. We conclude that along with amphipathicity, a stable secondary structure that can promote/initiate membrane anchoring is key in determining membrane destabilization potential of these AMPs. Our findings are a step towards understanding the role of specific residues in antimicrobial activity of BMAP-28(1-18), which will facilitate design of smaller, cost-effective therapeutics and would also help prediction algorithms to expedite screening out variants of the parent peptide with greater accuracy.
Comparing cryo-EM structures.
Venue: Journal of structural biology

Electron cryo-microscopy (cryo-EM), is a rapidly emerging method for the determination of macromolecular complexes with an expanding presence in both the scientific literature and public data archives. Indeed, its role in moving biochemistry into a new era was recognized by the 2017 Nobel Prize in Chemistry to Jacques Dubochet, Joachim Frank, and Richard Henderson (nobelprize.org/prizes/chemistry/2017). EM Data Bank (EMDB) now holds more than 6500 cryo-EM density maps, and Protein Data Bank (PDB) holds more than 2500 structure coordinates, contributed by the growing cryo-EM community (Patwardhan and Lawson, 2016).
Structural insights into the specificity and catalytic mechanism of mycobacterial nucleotide pool sanitizing enzyme MutT2.
Venue: Journal of structural biology

Mis-incorporation of modified nucleotides, such as 5-methyl-dCTP or 8-oxo-dGTP, in DNA can be detrimental to genomic integrity. MutT proteins are sanitization enzymes which function by hydrolyzing such nucleotides and regulating the pool of free nucleotides in the cytoplasm. Mycobacterial genomes have a set of four MutT homologs, namely, MutT1, MutT2, MutT3 and MutT4. Mycobacterial MutT2 hydrolyzes 5 m-dCTP and 8-oxo-dGTP to their respective monophosphate products. Additionally, it can hydrolyze canonical nucleotides dCTP and CTP, with a suggested role in sustaining their optimal levels in the nucleotide pool. The structures of M. smegmatis MutT2 and its complexes with cytosine derivatives have been determined at resolutions ranging from 1.10 Å to 1.73 Å. The apo enzyme and its complexes with products (dCMP, CMP and 5 m-dCMP) crystallize in space group P21212, while those involving substrates (dCTP, CTP and 5 m-dCTP) crystallize in space group P21. The molecule takes an α/β/α sandwich fold arrangement, as observed in other MutT homologs. The nucleoside moiety of the ligands is similarly located in all the complexes, while the location of the remaining tail exhibits variability. This is the first report of a MutT2-type protein in complex with ligands. A critical interaction involving Asp116 confers the specificity of the enzyme towards cytosine moieties. A conserved set of enzyme-ligand interactions along with concerted movements of important water molecules provide insights into the mechanism of action.
Using Scipion for stream image processing at Cryo-EM facilities
Venue: Journal of structural biology

Three dimensional electron microscopy is becoming a very data-intensive field in which vast amounts of experimental images are acquired at high speed. To manage such large-scale projects, we had previously developed a modular workflow system called Scipion (de la Rosa-Trevín et al., 2016). We present here a major extension of Scipion that allows processing of EM images while the data is being acquired. This approach helps to detect problems at early stages, saves computing time and provides users with a detailed evaluation of the data quality before the acquisition is finished. At present, Scipion has been deployed and is in production mode in seven Cryo-EM facilities throughout the world.
Crystal structure of a biliverdin-bound phycobiliprotein: Interdependence of oligomerization and chromophorylation.
Venue: Journal of structural biology

Small, ultra-red fluorescence protein (smURFP) introduces the non-native biliverdin (BV) chromophore to phycobiliproteins (PBPs), allowing them to be used as transgenic labels for in vivo mammalian imaging. Presently, no structural information exists for PBPs bound to the non-native BV chromophore, which limits the further development of smURFP and related proteins as imaging labels or indicators. Here we describe the first crystal structure of a PBP bound to BV. The structures of smURFP-Y56R with BV and smURFP-Y56F without BV reveal unique oligomerization interfaces different from those in wild-type PBPs bound to native chromophores. Our structures suggest that the oligomerization interface affects the BV binding site, creating a link between oligomerization and chromophorylation that we confirmed through site-directed mutagenesis and that may help guide efforts to improve the notorious chromophorylation of smURFP and other PBPs engineered to bind BV.
Tailored disorder in calcite organization in tergite cuticle of the supralittoral isopod Tylos europaeus Arcangeli, 1938.
Venue: Journal of structural biology

The crustacean cuticle forms skeletal elements consisting of chitin-protein fibrils reinforced by amorphous and crystalline calcium carbonate and phosphate minerals. The edges of skeletal elements are of particular interest. They are subject to repeated strain and stress because they form transitions to the arthrodial membranes connecting them. These allow for relative movements of skeletal elements. In this study, we investigate structure, chemical composition, mineral organization and local mechanical properties of the anterior and posterior edges of the tergite cuticle in the conglobating beach isopod Tylos europaeus and compare these with the protective dorsal region of the tergites. The distribution of mineral phases at the edges resembles that of dorsal regions of the tergites. At the transition with the unmineralized arthrodial membrane the calcite containing distal exocuticle is replaced by epicuticular material and the subjacent cuticular layers containing amorphous calcium carbonate become enriched with amorphous calcium phosphate. At the edges, the local elastic modulus and hardness values are significantly lower compared to dorsal regions of the tergite cuticle, for both, the calcite and the amorphous mineral containing layers. The calcite within the tergite cuticle is assembled in different texture patterns: (i) almost random co-orientation, (ii) almost single crystalline calcite, and (iii) a graded organization. Calcite organization and co-orientation strength is highly variable, not only on very few tens of micrometres, but also between regions with different skeletal functionality. Our results show that besides structure and composition, patterns of calcite organization contribute to the hierarchical architecture and functionality of biological composites.
Map challenge: Analysis using a pair comparison method based on Fourier shell correlation.
Venue: Journal of structural biology

This document presents the analysis performed over the Map Challenge dataset using a new algorithm which we refer to as Pair Comparison Method. The new algorithm, which is described in detail in the text, is able to sort reconstructions based on a figure of merit and assigns a level of significance to the sorting. That is, it shows how likely the sorting is due to chance or if it reflects real differences.
DNP NMR of biomolecular assemblies.
Venue: Journal of structural biology

Dynamic Nuclear Polarization (DNP) is an effective approach to alleviate the inherently low sensitivity of solid-state NMR (ssNMR) under magic angle spinning (MAS) towards large-sized multi-domain complexes and assemblies. DNP relies on a polarization transfer at cryogenic temperatures from unpaired electrons to adjacent nuclei upon continuous microwave irradiation. This is usually made possible via the addition in the sample of a polarizing agent. The first pioneering experiments on biomolecular assemblies were reported in the early 2000s on bacteriophages and membrane proteins. Since then, DNP has experienced tremendous advances, with the development of extremely efficient polarizing agents or with the introduction of new microwaves sources, suitable for NMR experiments at very high magnetic fields (currently up to 900 MHz). After a brief introduction, several experimental aspects of DNP enhanced NMR spectroscopy applied to biomolecular assemblies are discussed. Recent demonstration experiments of the method on viral capsids, the type III and IV bacterial secretion systems, ribosome and membrane proteins are then described.
Structural characterization of the sporulation protein GerM from Bacillus subtilis.
Venue: Journal of structural biology

The Gram-positive bacterium Bacillus subtilis responds to starvation by entering a morphological differentiation process leading to the formation of a highly resistant spore. Early in the sporulation process, the cell asymmetrically divides into a large compartment (the mother cell) and a smaller one (the forespore), which will maturate into a resistant spore. Proper development of the forespore requires the assembly of a multiprotein complex called the SpoIIIA-SpoIIQ complex or "A-Q complex". This complex involves the forespore protein SpoIIQ and eight mother cell proteins (SpoIIIAA to SpoIIIAH), many of which share structural similarities with components of specialized secretion systems and flagella found in Gram-negative bacteria. The assembly of the A-Q complex across the two membranes that separate the mother cell and forespore was recently shown to require GerM. GerM is a lipoprotein composed of two GerMN domains, a family of domains with unknown function. Here, we report X-ray crystallographic structures of the first GerMN domain of GerM at 1.0 Å resolution, and of the soluble domain of GerM (the tandem of GerMN domains) at 2.1 Å resolution. These structures reveal that GerMN domains can adopt distinct conformations and that the core of these domains display structural similarities with ring-building motifs found in components of specialized secretion system and in SpoIIIA proteins. This work provides an additional piece towards the structural characterization of the A-Q complex.
Fast multiscale reconstruction for Cryo-EM☆
Venue: Journal of structural biology

We present a multiscale reconstruction framework for single-particle analysis (SPA). The representation of three-dimensional (3D) objects with scaled basis functions permits the reconstruction of volumes at any desired scale in the real-space. This multiscale approach generates interesting opportunities in SPA for the stabilization of the initial volume problem or the 3D iterative refinement procedure. In particular, we show that reconstructions performed at coarse scale are more robust to angular errors and permit gains in computational speed. A key component of the proposed iterative scheme is its fast implementation. The costly step of reconstruction, which was previously hindering the use of advanced iterative methods in SPA, is formulated as a discrete convolution with a cost that does not depend on the number of projection directions. The inclusion of the contrast transfer function inside the imaging matrix is also done at no extra computational cost. By permitting full 3D regularization, the framework is by itself a robust alternative to direct methods for performing reconstruction in adverse imaging conditions (e.g., heavy noise, large angular misassignments, low number of projections). We present reconstructions obtained at different scales from a dataset of the 2015/2016 EMDataBank Map Challenge. The algorithm has been implemented in the Scipion package.
Direct evidence supporting the existence of a helical dislocation in protofilament packing in the intermediate filaments of oxidized trichocyte keratin.
Venue: Journal of structural biology

The X-ray diffraction patterns of quill and hair, as well as other trichocyte keratin appendages, contain meridional reflections that can be indexed on an axial repeat of 470 Å. Unusually, however, many of the expected orders are not observed. A possible explanation, proposed by Fraser and MacRae (1983), was that the intermediate filaments (IF) that constitute the fibrillar component of the filament/matrix texture consist of 4-chain protofilaments arranged on a surface lattice subject to a helical dislocation. The radial projection of the resulting 8-protofilament ribbon was defined in terms of a two-dimensional unit cell characterized by vectors (a, b) with axial projections za ∼ 74 Å and zb ∼ 198 Å. This situation resembles that found in microtubules, where helical dislocations in subunit packing are also encountered, leading to a so-called "seam" along their length (Metoz and Wade, 1997). In keratin, however, the protofilaments are helical so the seam is inclined to the axis of the IF. Here we report details of the Patterson function that provides independent evidence for both the helical dislocation and the dimensions of the surface lattice. In addition, the observed meridional X-ray amplitudes have been compared with those predicted by various models of the axial distribution of electron density. A new model, adapted from one previously proposed, fits the data significantly better than has heretofore proved possible. An interpretation of the model in terms of either specific keratin-associated-protein (KAP) binding or the retention of IF symmetry by a portion of the head and/or tail domains is suggested.
Structural modulation of a periplasmic sugar-binding protein probes into its evolutionary ancestry.
Venue: Journal of structural biology

Substrate-binding proteins (SBPs) are periplasmic proteins consisting of two α/β domains joined by a hinge region with specificity towards cognate ligands. Based on three-dimensional fold, sugar-specific SBPs have been classified into cluster B and cluster D-I. The analysis of sequences and structures of sugar-binding pocket of cluster D-I SBPs revealed the presence of extra residues on two loops (L1, L2) and a helix (H1) in few members of this family, that binds specifically to monosaccharides. Presence of conserved histidine in L2 and tryptophan in H1 can be considered as the identity marks for the cluster D-I monosaccharide-binding SBPs. A glucose binding protein (ppGBP) from Pseudomonas putida CSV86 was found to contain a structural fold similar to oligosaccharide-binding cluster D-I SBPs, but functionally binds to only glucose due to constriction of its binding pocket mainly by L2 (375-382). ppGBP with partial deletion of L2 (ppGBPΔL2) was created, crystallized and biochemical characterization was performed. Compared to wild type ppGBP, the ppGBPΔL2 structure showed widening of the glucose-binding pocket with ∼80% lower glucose binding. Our results show that the substrate specificity of SBPs can be altered by modulating the size of the binding pocket. Based on this, we propose a sub classification of cluster D-I SBPs into (i) cluster D-I(a)-monosaccharide-binding SBPs and (ii) cluster D-I(b)-oligosaccharide-binding SBPs. This study also provides the direct structural and functional correlation indicating that divergence of proteins may occur through insertions or deletions of sequences in the already existing SBPs leading to evolution at the functional level.
Automation and assessment of de novo modeling with Pathwalking in near atomic resolution cryoEM density maps.
Venue: Journal of structural biology

With the rapidly growing number of macromolecular structures solved to near-atomic resolution using electron cryomicroscopy (cryoEM), map interpretation and model building directly from the density without the use of structural templates has become increasingly important. As part of the 2015/2016 Map and Model Challenge, we attempted to assess our latest de novo modeling tool, Pathwalking, in terms of performance and usability, as well as identify areas for future improvements. In total, we applied Pathwalking to six density maps between 3 and 4.5 Å resolution selected from the challenge data sets. In five of the six cases, Pathwalking was able to accurately determine the protein fold and in three of these cases, the final all atom model had less than 1.6 Å RMSD when compared to the known structure. Model building and refinement was nearly completely automated, used default parameters and took less than 30 min to complete a refined all atom model. A direct outgrowth of this work was a more streamlined automated command line Pathwalking utility, as well as a novel sequence assignment and optimization routine, which attempts to register sidechain density with expected side chain volume. In total, Pathwalking offers a nearly complete, robust and efficient method for constructing atomistic protein structures directly from a density map without the aid of a template.
Assessing the quality of single particle reconstructions by atomic model building.
Venue: Journal of structural biology

The 2015/2016 Map Challenge challenged cryo-EM practitioners to process a series of publicly available cryo-EM datasets. As part of the challenge, metrics needed to be developed to assess and compare the quality of the different map submissions. The most common metric for assessing maps is determining the resolution by Fourier shell correlation (FSC), but there are well known instances where the resolution can be misleading. In this manuscript, we present a new approach for assessing the quality of a map by determining the map "modelability" rather than on resolution. We used the automated map tracing and modeling algorithms in Rosetta to generate populations of models, and then compared the populations between different map entries by the Rosetta score, RMSD to a reference model provided by the map challenge, and by pair-wise RMSDs between different models in the population. These metrics were used to determine statistically significant rankings for the map challengers for each dataset. The rankings revealed inconsistencies between the resolution by FSC, emphasized the importance of the interplay between number of particles contributing to a map and map quality, and revealed the importance of software familiarity on single particle reconstruction results. However, because multiple variables changed between map entries, it was challenging to derive best practices from the map challenge results.
Advances in instrumentation and methodology for solid-state NMR of biological assemblies.
Venue: Journal of structural biology

Many advances in instrumentation and methodology have furthered the use of solid-state NMR as a technique for determining the structures and studying the dynamics of molecules involved in complex biological assemblies. Solid-state NMR does not require large crystals, has no inherent size limit, and with appropriate isotopic labeling schemes, supports solving one component of a complex assembly at a time. It is complementary to cryo-EM, in that it provides local, atomic-level detail that can be modeled into larger-scale structures. This review focuses on the development of high-field MAS instrumentation and methodology; including probe design, benchmarking strategies, labeling schemes, and experiments that enable the use of quadrupolar nuclei in biomolecular NMR. Current challenges facing solid-state NMR of biological assemblies and new directions in this dynamic research area are also discussed.
Polyserine repeats promote coiled coil-mediated fibril formation and length-dependent protein aggregation.
Venue: Journal of structural biology

Short polyserine (polyS) repeats are frequently found in proteins and longer ones are produced in neurological disorders such as Huntington disease (HD) owing to translational frameshifting or non-ATG-dependent translation, together with polyglutamine (polyQ) and polyalanine (polyA) repeats, forming intracellular aggregates. However, the physiological and pathological structures of polyS repeats are not clearly understood. Early studies highlighted their structural versatility, similar to other homopolymers whose conformation is influenced by the surrounding protein context. As polyS stretches are frequently near polyQ and polyA repeats, which can be part of coiled coil (CC) structures, and the frameshift-derived polyS repeats in HD directly flank CC heptads important for aggregation, we investigate here the structural and aggregation properties of polyS in the context of CC structures. We have taken advantage of peptide models, previously used to study polyQ and polyA in CCs, in which we inserted polyS repeats of variable length and studied them in comparison with polyQ and polyA peptides. We found that polyS repeats promote CC-mediated polymerization and fibrillization as revealed by circular dichroism, chemical crosslinking, and atomic force microscopy. Furthermore, they promote CC-based, length-dependent intracellular aggregation, which is negligible with 7 and widespread with 49 serines. These findings show that polyS repeats can participate in the formation of CCs, as previously found for polyQ and polyA, conferring to peptides distinctive structural properties with aggregation kinetics that are intermediate between those of polyA and polyQ CCs, and contribute to an overall structural definition of the pathophysiogical roles of homopolymeric repeats in CC structures.
New software tools in EMAN2 inspired by EMDatabank map challenge.
Venue: Journal of structural biology

EMAN2 is an extensible software suite with complete workflows for performing high-resolution single particle analysis, 2-D and 3-D heterogeneity analysis, and subtomogram averaging, among other tasks. Participation in the recent CryoEM Map Challenge sponsored by the EMDatabank led to a number of significant improvements to the single particle analysis process in EMAN2. A new convolutional neural network particle picker was developed, which dramatically improves particle picking accuracy for difficult data sets. A new particle quality metric capable of accurately identifying "bad" particles with a high degree of accuracy, no human input, and a negligible amount of additional computation, has been introduced, and this now serves as a replacement for earlier human-biased methods. The way 3-D single particle reconstructions are filtered has been altered to be more comparable to the filter applied in several other popular software packages, dramatically improving the appearance of sidechains in high-resolution structures. Finally, an option has been added to perform local resolution-based iterative filtration, resulting in local resolution improvements in many maps.