nils-herrmann/clean_scirepeval huggingface dataset:
A new HIV-1 Rev structure optimizes interaction with target RNA (RRE) for nuclear export.
Venue: Journal of structural biology

HIV-1 Rev mediates the nuclear export of unspliced and partially-spliced viral transcripts for the production of progeny genomes and structural proteins. In this process, four (or more) copies of Rev assemble onto a highly-structured 351-nt region in such viral transcripts, the Rev response element (RRE). How this occurs is not known. The Rev assembly domain has a helical-hairpin structure which associates through three (A-A, B-B and C-C) interfaces. The RRE has the topology of an upper-case letter A, with the two known Rev binding sites mapping onto the legs of the A. We have determined a crystal structure for the Rev assembly domain at 2.25 Å resolution, without resort to either mutations or chaperones. It shows that B-B dimers adopt an arrangement reversed relative to that previously reported, and join through a C-C interface to form tetramers. The new subunit arrangement shows how four Rev molecules can assemble on the two sites on the RRE to form the specificity checkpoint, and how further copies add through A-A interactions. Residues at the C-C interface, specifically the Pro31-Trp45 axis, are a potential target for intervention.
Amyloid structure of high-order assembly of Leucine-rich amelogenin revealed by solid-state NMR.
Venue: Journal of structural biology

High-order assemblies of amelogenin, the major protein in enamel protein matrix, are believed to act as the template for enamel mineral formation. The Leucine-rich amelogenin (LRAP) is a natural splice-variant of amelogenin, a functional protein in vivo, containing conserved domains of amelogenin. In this work, we showed LRAP aggregates hierarchically into assemblies with various sizes including scattered beads, beads-on-a-string and gel-like precipitations in the presence of both calcium and phosphate ions. Solid-state NMR combined with X-ray diffraction and microscopic techniques, was applied to give a picture of LRAP self-assemblies at the atomic level. Our results, for the first time, confirmed LRAP assemblies with different sizes all contained a consistent rigid segment with β-sheet secondary structure (residues 12-27) and the β-sheet segment would further assemble into amyloid-like structures.
Structure and electrochemistry of proteins harboring iron-sulfur clusters of different nuclearities. Part III. [4Fe-4S], [3Fe-4S] and [2Fe-2S] iron-sulfur proteins.
Venue: Journal of structural biology

A systematic rationalization of the hundreds of proteins harboring iron-sulfur clusters and able to exhibit the most diverse biological functions is missing. In this picture we have already reviewed structure/electrochemistry of metalloproteins expressing single types of iron-sulfur centres [namely, {Fe(Cys)4}, {[Fe2S2](Cys)4}, {[Fe2S2](Cys)3(X)} (X = Asp, Arg, His), {[Fe2S2](Cys)2(His)2}, {[Fe3S4](Cys)3}, {[Fe4S4](Cys)4} and {[Fe4S4](SγCys)3(nonthiolate ligand)}] and their synthetic analogs. Recently we are focussing on structure/electrochemistry of metalloproteins containing iron-sulfur centres of different nuclearities. Having started such a subject with proteins harboring [4Fe-4S] and [2Fe-2S] (Zanello, 2017c) as well as [4Fe-4S] and [3Fe-4S] (Zanello, in press) clusters, we now provide the state of art of proteins harboring [4Fe-4S], [3Fe-4S] and [2Fe-2S] clusters, a subject that resulted strictly limited to enzymes active in the respiratory Complex II.
Blind estimation of DED camera gain in Electron Microscopy.
Venue: Journal of structural biology

The introduction of Direct Electron Detector (DED) videos in the Electron Microscope field has boosted Single Particle Analysis to a point in which it is currently considered to be a key technique in Structural Biology. In this article we introduce an approach to estimate the DED camera gain at each pixel from the movies themselves. This gain is needed to have the set of recorded frames into a coherent gray level range, homogeneous over the whole image. The algorithm does not need any other input than the DED movie itself, being capable of providing an estimate of the camera gain image, helping to identify dead pixels and cases of incorrectly calibrated cameras. We propose the algorithm to be used either to validate the experimentally acquired gain image (for instance, to follow its possible change over time) or to verify that there is no residual gain image after experimentally correcting for the camera gain. We show results for a number of DED camera models currently in use (DE, Falcon II, Falcon 3, and K2).
Improved sample dispersion in cryo-EM using "perpetually-hydrated" graphene oxide flakes.
Venue: Journal of structural biology

For many macromolecular complexes, the inability to uniformly disperse solubilized specimen particles within vitreous ice films precludes their analysis by cryo-electron microscopy (cryo-EM). Here, we introduce a sample preparation process using "perpetually-hydrated" graphene oxide flakes as particle support films, and report vastly improved specimen dispersion. Furthermore, we provide evidence that the presence of graphene oxide flakes in vitreous ice results in a significant reduction in electron beam-induced specimen decomposition. The new method introduced in this study incorporates hydrated graphene oxide flakes into a standard sample preparation regime, without the need for additional tools or devices, making it a cost-effective and easily adoptable alternative to currently available sample preparation approaches.
Structure of a prokaryotic SEFIR domain reveals two novel SEFIR-SEFIR interaction modes.
Venue: Journal of structural biology

SEFIR domain-containing proteins are crucial for mammalian adaptive immunity. As a unique intracellular signaling domain, the SEFIR-SEFIR interactions mediate physical protein-protein interactions in the immune signaling network, especially the IL-17- and IL-25-mediated pathways. However, due to the lack of structural information, the detailed molecular mechanism for SEFIR-SEFIR assembly remains unclear. In the present study, we solved the crystal structures of a prokaryotic SEFIR domain from Bacillus cereus F65185 (BcSEFIR), where the SEFIR domain is located at the N terminus. The structure of BcSEFIR revealed two radically distinct SEFIR-SEFIR interaction modes. In the asymmetric form, the C-terminal tail of one SEFIR binds to the helix αA and βB-αB' segment of the other one, while in the symmetric form, the helices ηC and αE and the DE-segment compose the inter-protomer interface. The C-terminal tail of BcSEFIR, critical for asymmetric interaction, is highly conserved among the SEFIR domains of Act1 orthologs from different species, in particular three absolutely conserved residues that constitute an EXXXXPP motif. In the symmetric interaction mode, the most significant contacts made by residues on helix αE are highly conserved in Act1 SEFIR domains, constituted an RLI/LXE motif. The two novel SEFIR-SEFIR interaction modes might explain the structural basis for SEFIR domain-mediated complex assembly in signaling pathways.
Baculovirus-driven protein expression in insect cells: A benchmarking study.
Venue: Journal of structural biology

Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E. coli transposition-based recombination bacmid technology (e.g. Bac-to-Bac®) or homologous recombination transfection within insect cells (e.g. flashBAC™). Limited data is presented in the literature to benchmark the protocols used for these baculovirus vectors to facilitate the selection of a system for optimal production of target proteins. Taking advantage of the Protein Production and Purification Partnership in Europe (P4EU) scientific network, a benchmarking initiative was designed to compare the diverse protocols established in thirteen individual laboratories. This benchmarking initiative compared the expression of four selected intracellular proteins (mouse Dicer-2, 204 kDa; human ABL1 wildtype, 126 kDa; human FMRP, 68 kDa; viral vNS1-H1, 76 kDa). Here, we present the expression and purification results on these proteins and highlight the significant differences in expression yields obtained using different commercially-packaged baculovirus vectors. The highest expression level for difficult-to-express intracellular protein candidates were observed with the EmBacY baculovirus vector system.
Solution structure of an archaeal DUF61 family protein SSO0941 encoded by a gene in the operon of box C/D RNA protein complexes.
Venue: Journal of structural biology

Domain of unknown function 61 (DUF61) family proteins widely exist in archaea and the genes of DUF61 proteins in crenarchaea are in an operon containing two genes of box C/D RNA protein complexes. Here we report the solution NMR structure of DUF61 family member protein SSO0941, from the hyperthermophilic archaeon Sulfolobus solfataricus. SSO0941 has a rigid core structure and flexible N- and C-terminal regions as well as a negatively-charged independent C-terminal helix. The core structure consists of N- and C-terminal subdomains, in which the C-terminal subdomain shows significant structural similarity with several nucleic acid binding proteins. The structure of SSO0941 is the first representative structure of DUF61 family proteins.
Gaussian-input Gaussian mixture model for representing density maps and atomic models.
Venue: Journal of structural biology

A new Gaussian mixture model (GMM) has been developed for better representations of both atomic models and electron microscopy 3D density maps. The standard GMM algorithm employs an EM algorithm to determine the parameters. It accepted a set of 3D points with weights, corresponding to voxel or atomic centers. Although the standard algorithm worked reasonably well; however, it had three problems. First, it ignored the size (voxel width or atomic radius) of the input, and thus it could lead to a GMM with a smaller spread than the input. Second, the algorithm had a singularity problem, as it sometimes stopped the iterative procedure due to a Gaussian function with almost zero variance. Third, a map with a large number of voxels required a long computation time for conversion to a GMM. To solve these problems, we have introduced a Gaussian-input GMM algorithm, which considers the input atoms or voxels as a set of Gaussian functions. The standard EM algorithm of GMM was extended to optimize the new GMM. The new GMM has identical radius of gyration to the input, and does not suddenly stop due to the singularity problem. For fast computation, we have introduced a down-sampled Gaussian functions (DSG) by merging neighboring voxels into an anisotropic Gaussian function. It provides a GMM with thousands of Gaussian functions in a short computation time. We also have introduced a DSG-input GMM: the Gaussian-input GMM with the DSG as the input. This new algorithm is much faster than the standard algorithm.
Structural basis for partition of the cyclodipeptide synthases into two subfamilies.
Venue: Journal of structural biology

Cyclodipeptide synthases (CDPSs) use two aminoacyl-tRNAs to catalyze the formation of two peptide bonds leading to cyclodipeptides that can be further used for the synthesis of diketopiperazines. It was shown that CDPSs fall into two subfamilies, NYH and XYP, characterized by the presence of specific sequence signatures. However, current understanding of CDPSs only comes from studies of enzymes from the NYH subfamily. The present study reveals the crystal structures of three CDPSs from the XYP subfamily. Comparison of the XYP and NYH enzymes shows that the two subfamilies mainly differ in the first half of their Rossmann fold. This gives a structural basis for the partition of CDPSs into two subfamilies. Despite these differences, the catalytic residues adopt similar positioning regardless of the subfamily suggesting that the XYP and NYH motifs correspond to two structural solutions to facilitate the reactivity of the catalytic serine residue.
αCGRP, another amyloidogenic member of the CGRP family.
Venue: Journal of structural biology

The Calcitonin-gene related peptide (CGRP) family is a group of peptide hormones, which consists of IAPP, calcitonin, adrenomedullin, intermedin, αCGRP and βCGRP. IAPP and calcitonin have been extensively associated with the formation of amyloid fibrils, causing Type 2 Diabetes and Medullary Thyroid Carcinoma, respectively. In contrast, the potential amyloidogenic properties of αCGRP still remain unexplored, although experimental trials have indicated its presence in deposits, associated with the aforementioned disorders. Therefore, in this work, we investigated the amyloidogenic profile of αCGRP, a 37-residue-long peptide hormone, utilizing both biophysical experimental techniques and Molecular Dynamics simulations. These efforts unravel a novel amyloidogenic member of the CGRP family and provide insights into the mechanism underlying the αCGRP polymerization.
Interplay between membrane curvature and protein conformational equilibrium investigated by solid-state NMR.
Venue: Journal of structural biology

Many membrane proteins sense and induce membrane curvature for function, but structural information about how proteins modulate their structures to cause membrane curvature is sparse. We review our recent solid-state NMR studies of two virus membrane proteins whose conformational equilibrium is tightly coupled to membrane curvature. The influenza M2 proton channel has a drug-binding site in the transmembrane (TM) pore. Previous chemical shift data indicated that this pore-binding site is lost in an M2 construct that contains the TM domain and a curvature-inducing amphipathic helix. We have now obtained chemical shift perturbation, protein-drug proximity, and drug orientation data that indicate that the pore-binding site is restored when the full cytoplasmic domain is present. This finding indicates that the curvature-inducing amphipathic helix distorts the TM structure to interfere with drug binding, while the cytoplasmic tail attenuates this effect. In the second example, we review our studies of a parainfluenza virus fusion protein that merges the cell membrane and the virus envelope during virus entry. Chemical shifts of two hydrophobic domains of the protein indicate that both domains have membrane-dependent backbone conformations, with the β-strand structure dominating in negative-curvature phosphatidylethanolamine (PE) membranes. 31P NMR spectra and 1H-31P correlation spectra indicate that the β-strand-rich conformation induces saddle-splay curvature to PE membranes and dehydrates them, thus stabilizing the hemifusion state. These results highlight the indispensable role of solid-state NMR to simultaneously determine membrane protein structures and characterize the membrane curvature in which these protein structures exist.
Exploring applications of crowdsourcing to cryo-EM
Venue: Journal of structural biology

Extraction of particles from cryo-electron microscopy (cryo-EM) micrographs is a crucial step in processing single-particle datasets. Although algorithms have been developed for automatic particle picking, these algorithms generally rely on two-dimensional templates for particle identification, which may exhibit biases that can propagate artifacts through the reconstruction pipeline. Manual picking is viewed as a gold-standard solution for particle selection, but it is too time-consuming to perform on data sets of thousands of images. In recent years, crowdsourcing has proven effective at leveraging the open web to manually curate datasets. In particular, citizen science projects such as Galaxy Zoo have shown the power of appealing to users’ scientific interests to process enormous amounts of data. To this end, we explored the possible applications of crowdsourcing in cryo-EM particle picking, presenting a variety of novel experiments including the production of a fully annotated particle set from untrained citizen scientists. We show the possibilities and limitations of crowdsourcing particle selection tasks, and explore further options for crowdsourcing cryo-EM data processing.
Coiled-coil oligomerization controls localization of the plasma membrane REMORINs.
Venue: Journal of structural biology

REMORINs are nanodomain-organized proteins located in the plasma membrane and involved in cellular responses in plants. The dynamic assembly of the membrane nanodomains represents an essential tool of the versatile membrane barriers to control and modulate cellular functions. Nevertheless, the assembly mechanisms and protein organization strategies of nanodomains are poorly understood and many structural aspects are difficult to visualize. Using an ensemble of biophysical approaches, including solid-state nuclear magnetic resonance, cryo-electron microscopy and in vivo confocal imaging, we provide first insights on the role and the structural mechanisms of REMORIN trimerization. Our results suggest that the formation of REMORIN coiled-coil trimers is essential for membrane recruitment and promotes REMORIN assembly in vitro into long filaments by trimer-trimer interactions that might participate in nanoclustering into membrane domains in vivo.
An automated workflow for segmenting single adult cardiac cells from large-volume serial block-face scanning electron microscopy data.
Venue: Journal of structural biology

This paper presents a new algorithm to automatically segment the myofibrils, mitochondria and nuclei within single adult cardiac cells that are part of a large serial-block-face scanning electron microscopy (SBF-SEM) dataset. The algorithm only requires a set of manually drawn contours that roughly demarcate the cell boundary at routine slice intervals (every 50th, for example). The algorithm correctly classified pixels within the single cell with 97% accuracy when compared to manual segmentations. One entire cell and the partial volumes of two cells were segmented. Analysis of segmentations within these cells showed that myofibrils and mitochondria occupied 47.5% and 51.6% on average respectively, while the nuclei occupy 0.7% of the cell for which the entire volume was captured in the SBF-SEM dataset. Mitochondria clustering increased at the periphery of the nucleus region and branching points of the cardiac cell. The segmentations also showed high area fraction of mitochondria (up to 70% of the 2D image slice) in the sub-sarcolemmal region, whilst it was closer to 50% in the intermyofibrillar space. We finally demonstrate that our segmentations can be turned into 3D finite element meshes for cardiac cell computational physiology studies. We offer our large dataset and MATLAB implementation of the algorithm for research use at www.github.com/CellSMB/sbfsem-cardiac-cell-segmenter/. We anticipate that this timely tool will be of use to cardiac computational and experimental physiologists alike who study cardiac ultrastructure and its role in heart function.
Combining Rosetta with molecular dynamics (MD): A benchmark of the MD-based ensemble protein design.
Venue: Journal of structural biology

Computational protein design is a set of procedures for computing amino acid sequences that will fold into a specified structure. Rosetta Design, a commonly used software for protein design, allows for the effective identification of sequences compatible with a given backbone structure, while molecular dynamics (MD) simulations can thoroughly sample near-native conformations. We benchmarked a procedure in which Rosetta design is started on MD-derived structural ensembles and showed that such a combined approach generates 20-30% more diverse sequences than currently available methods with only a slight increase in computation time. Importantly, the increase in diversity is achieved without a loss in the quality of the designed sequences assessed by their resemblance to natural sequences. We demonstrate that the MD-based procedure is also applicable to de novo design tasks started from backbone structures without any sequence information. In addition, we implemented a protocol that can be used to assess the stability of designed models and to select the best candidates for experimental validation. In sum our results demonstrate that the MD ensemble-based flexible backbone design can be a viable method for protein design, especially for tasks that require a large pool of diverse sequences.
Structure and electrochemistry of proteins harboring iron-sulfur clusters of different nuclearities. Part II. [4Fe-4S] and [3Fe-4S] iron-sulfur proteins.
Venue: Journal of structural biology

In the context of the plethora of proteins harboring iron-sulfur clusters we have already reviewed structure/electrochemistry of metalloproteins expressing single types of iron-sulfur clusters (namely: {Fe(Cys)4}, {[Fe2S2](Cys)4}, {[Fe2S2](Cys)3(X)} (X = Asp, Arg, His), {[Fe2S2](Cys)2(His)2}, {[Fe3S4](Cys)3}, {[Fe4S4](Cys)4} and {[Fe4S4](SγCys)3(nonthiolate ligand)} cores) and their synthetic analogs. More recently we are focussing on structure/electrochemistry of metalloproteins harboring iron-sulfur centres of different nuclearities. Having started such a subject with proteins harboring [4Fe-4S] and [2Fe-2S] clusters, we now depict the state of art of proteins containing [4Fe-4S] and [3Fe-4S] clusters.
A complete structural characterization of the desferrioxamine E biosynthetic pathway from the fire blight pathogen Erwinia amylovora.
Venue: Journal of structural biology

The Gram-negative bacterium Erwinia amylovora is the etiological agent of fire blight, a devastating disease which affects Rosaceae such as apple, pear and quince. The siderophore desferrioxamine E plays an important role in bacterial pathogenesis by scavenging iron from the host. DfoJ, DfoA and DfoC are the enzymes responsible for desferrioxamine production starting from lysine. We have determined the crystal structures of each enzyme in the desferrioxamine E pathway and demonstrate that the biosynthesis involves the concerted action of DfoJ, followed by DfoA and lastly DfoC. These data provide the first crystal structures of a Group II pyridoxal-dependent lysine decarboxylase, a cadaverine monooxygenase and a desferrioxamine synthetase. DfoJ is a homodimer made up of three domains. Each monomer contributes to the completion of the active site, which is positioned at the dimer interface. DfoA is the first structure of a cadaverine monooxygenase. It forms homotetramers whose subunits are built by two domains: one for FAD and one for NADP+ binding, the latter of which is formed by two subdomains. We propose a model for substrate binding and the role of residues 43-47 as gate keepers for FAD binding and the role of Arg97 in cofactors turnover. DfoC is the first structure of a desferrioxamine synthetase and the first of a multi-enzyme siderophore synthetase coupling an acyltransferase domain with a Non-Ribosomal Peptide Synthetase (NRPS)-Independent Siderophore domain (NIS).