nils-herrmann/clean_scirepeval huggingface dataset:
Study of the interactions between the key spore coat morphogenetic proteins CotE and SpoVID.
Venue: Journal of structural biology

The capability of Bacillus subtilis spores to withstand extreme environmental conditions is thought to be conferred especially by their outermost proteinaceous protective layer, called the spore coat. Of the over 70 proteins that form the spore coat, only a small subset of them affect its morphogenesis, they are referred to as morphogenetic proteins. In this study we investigated the interaction between two spore coat morphogenetic proteins SpoVID and CotE. SpoVID is involved in the process of spore surface encirclement by individual coat proteins, these include CotE, which controls the assembly of the outer coat layer. Both proteins were proposed to be recruited to a common protein scaffold, but their direct association has not been previously shown. Here we studied the interactions between CotE and SpoVID in vitro for the first time by using molecule recognition force spectroscopy, which allows the detection of piconewton forces between conjugated biological pairs and also facilitates the investigation of dynamic processes. The most probable CotE-CotE unbinding force was 49.4±0.1pN at a loading rate of 3.16×10³ pN/s while that of SpoVID-CotE was 26.5±0.6pN at a loading rate of 7.8×10² pN/s. We further analyzed the interactions with the bacterial two hybrid system and pull-down experiments, which also indicate that SpoVID interacts directly with CotE. In combination with the previously identified direct contacts among SpoIVA, SpoVID and SafA, our data imply that the physical association of key morphogenetic proteins forms a basic skeleton where other coat proteins could be attached.
In-house UV radiation-damage-induced phasing of selenomethionine-labeled protein structures.
Venue: Journal of structural biology

Selenomethionine labeling is the most common technique used in protein crystallography to derivatize recombinant proteins for experimental phasing using anomalous scattering at tunable synchrotron beamlines. Recently, it has been shown that UV radiation depletes electron density of selenium atoms of selenomethionine residues and that UV radiation-damage-induced phasing (equivalent to single isomorphous replacement) protocol can be applied to calculate experimental phases. Here we present the straightforward integration of a UV source with an in-house diffractometer. We show how this setup can extend the capabilities of a sealed tube X-ray generator and be used for experimental phasing of selenium-labeled proteins.
Crystal structures of acetate kinases from the eukaryotic pathogens Entamoeba histolytica and Cryptococcus neoformans.
Venue: Journal of structural biology

Acetate kinases (ACKs) are members of the acetate and sugar kinase/hsp70/actin (ASKHA) superfamily and catalyze the reversible phosphorylation of acetate, with ADP/ATP the most common phosphoryl acceptor/donor. While prokaryotic ACKs have been the subject of extensive biochemical and structural characterization, there is a comparative paucity of information on eukaryotic ACKs, and prior to this report, no structure of an ACK of eukaryotic origin was available. We determined the structures of ACKs from the eukaryotic pathogens Entamoeba histolytica and Cryptococcus neoformans. Each active site is located at an interdomain interface, and the acetate and phosphate binding pockets display sequence and structural conservation with their prokaryotic counterparts. Interestingly, the E. histolytica ACK has previously been shown to be pyrophosphate (PP(i))-dependent, and is the first ACK demonstrated to have this property. Examination of its structure demonstrates how subtle amino acid substitutions within the active site have converted cosubstrate specificity from ATP to PP(i) while retaining a similar backbone conformation. Differences in the angle between domains surrounding the active site suggest that interdomain movement may accompany catalysis. Taken together, these structures are consistent with the eukaryotic ACKs following a similar reaction mechanism as is proposed for the prokaryotic homologs.
An assignment of intrinsically disordered regions of proteins based on NMR structures.
Venue: Journal of structural biology

Intrinsically disordered proteins (IDPs) do not adopt stable three-dimensional structures in physiological conditions, yet these proteins play crucial roles in biological phenomena. In most cases, intrinsic disorder manifests itself in segments or domains of an IDP, called intrinsically disordered regions (IDRs), but fully disordered IDPs also exist. Although IDRs can be detected as missing residues in protein structures determined by X-ray crystallography, no protocol has been developed to identify IDRs from structures obtained by Nuclear Magnetic Resonance (NMR). Here, we propose a computational method to assign IDRs based on NMR structures. We compared missing residues of X-ray structures with residue-wise deviations of NMR structures for identical proteins, and derived a threshold deviation that gives the best correlation of ordered and disordered regions of both structures. The obtained threshold of 3.2Å was applied to proteins whose structures were only determined by NMR, and the resulting IDRs were analyzed and compared to those of X-ray structures with no NMR counterpart in terms of sequence length, IDR fraction, protein function, cellular location, and amino acid composition, all of which suggest distinct characteristics. The structural knowledge of IDPs is still inadequate compared with that of structured proteins. Our method can collect and utilize IDRs from structures determined by NMR, potentially enhancing the understanding of IDPs.
Disease-associated mutations in the coil 2B domain of human lamin A/C affect structural properties that mediate dimerization and intermediate filament formation.
Venue: Journal of structural biology

The lamin proteins are essential components of the nuclear lamina of eukaryotic cells, that are involved in a complex association mechanism to attain a functional supermolecular structure. Mutations of the lamin A/C gene are associated with several different neuromuscular diseases, and the detailed effect of disease-associated amino acid substitutions on the structure and stability of human lamin dimers is yet unknown. Here we present a structural and thermodynamic characterization by means of molecular dynamics simulations of the effect of pathological mutations (S326T, R331P, R331Q, E347K, E358K, M371K, and R377H) on the association of the coil 2B domains that mediate lamin A/C oligomerization. The structures attained during the simulations, along with the quantification of the contribution of each residue to the dimerization energies, support a lamin association mechanism mediated by homophilic intermolecular interactions promoted by dissociative conformational changes at distinct positions in the coiled coil. The pathogenic mutations can both increase or decrease the stability of lamin A/C dimers, and a possible correlation between the effect of the amino acid substitutions and disease onset and severity is presented.
Automated correlation of single particle tilt pairs for Random Conical Tilt and Orthogonal Tilt Reconstructions.
Venue: Journal of structural biology

One of the major methodological challenges in single particle electron microscopy is obtaining initial reconstructions which represent the structural heterogeneity of the dataset. Random Conical Tilt and Orthogonal Tilt Reconstruction techniques in combination with 3D alignment and classification can be used to obtain initial low-resolution reconstructions which represent the full range of structural heterogeneity of the dataset. In order to achieve statistical significance, however, a large number of 3D reconstructions, and, in turn, a large number of tilted image pairs are required. The extraction of single particle tilted image pairs from micrographs can be tedious and time-consuming, as it requires intensive user input even for semi-automated approaches. To overcome the bottleneck of manual selection of a large number of tilt pairs, we developed an algorithm for the correlation of single particle images from tilted image pairs in a fully automated and user-independent manner. The algorithm reliably correlates correct pairs even from noisy micrographs. We further demonstrate the applicability of the algorithm by using it to obtain initial references both from negative stain and unstained cryo datasets.
Characterization of β-domains in C-terminal fragments of TDP-43 by scanning tunneling microscopy.
Venue: Journal of structural biology

The TAR DNA-binding protein 43 (TDP-43) has been identified as a critical player in a range of neurodegenerative diseases, including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Recent discoveries demonstrate the important role of carboxyl-terminal fragments of TDP-43 in its proteinopathy. Herein, we report the characterization of β-domains in the C-terminal fragments of TDP-43 using scanning tunneling microscopy (STM). Careful comparison of the wild-type TDP-43 (Wt) and the three mutant TDP-43 peptides: an ALS-related mutant peptide: phosphorylated A315T mutant TDP-43 (A315T(p)) and two model peptides: A315T mutant TDP-43 (A315T), A315E mutant TDP-43 (A315E) reveals that A315T(p) has a longer core region of the β-domain than Wt. A315E possesses the longest core region of the β-domain and A315T(p) mutant TDP-43 has the second longest core region of the β-domain. The core regions of the β-domains for A315T and Wt TDP-43 have the same length. This observation provides a supportive evidence of a higher tendency in beta-sheet formation of A315T(p) containing TDP-43 fragment, and structural mechanism for the higher cytotoxicity and accelerated fibril formation of the A315T(p) mutation-containing TDP-43 peptide as compared with Wt TDP-43.
Poxvirus antagonism of innate immunity by Bcl-2 fold proteins.
Venue: Journal of structural biology

Poxviruses have evolved numerous mechanisms to evade host innate immunity. Sensory pathways that are activated by Toll-like and nucleotide receptors, as well as innate cell death pathways, are both targets of antagonism by viral proteins. Recent structural, biochemical and functional studies of poxvirus proteins have identified a family of α-helical proteins that adopt a Bcl-2 fold despite highly divergent polypeptide sequences from cellular proteins that regulate apoptosis. These newly identified proteins have assumed new roles in antagonism of NF-κB and interferon signaling pathways and interfere with the release of pro-inflammatory cytokines. Structures of isolated viral proteins and their complexes with cellular targets provide insight into the diverse ways that the Bcl-2 scaffold can be exploited for antagonism of host immunity.
Secondary osteon size and collagen/lamellar organization ("osteon morphotypes") are not coupled, but potentially adapt independently for local strain mode or magnitude.
Venue: Journal of structural biology

In bone, matrix slippage that occurs at cement lines of secondary osteons during loading is an important toughening mechanism. Toughness can also be enhanced by modifications in osteon cross-sectional size (diameter) for specific load environments; for example, smaller osteons in more highly strained "compression" regions vs. larger osteons in less strained "tension" regions. Additional osteon characteristics that enhance toughness are distinctive variations in collagen/lamellar organization (i.e., "osteon morphotypes"). Interactions might exist between osteon diameter and morphotype that represent adaptations for resisting deleterious shear stresses that occur at the cement line. This may be why osteons often have a peripheral ring (or "hoop") of highly oblique/transverse collagen. We hypothesized that well developed/distinct "hoops" are compensatory adaptations in cases where increased osteon diameter is mechanically advantageous (e.g., larger osteons in "tension" regions would have well developed/distinct "hoops" in order to resist deleterious consequences of co-existing localized shear stresses). We tested this hypothesis by determining if there are correlations between osteon diameters and strongly hooped morphotypes in "tension", "compression", and "neutral axis" regions of femora (chimpanzees, humans), radii (horse, sheep) and calcanei (horse, deer). The results reject the hypothesis-larger osteons are not associated with well developed/distinct "hoops", even in "tension regions" where the effect was expected to be obvious. Although osteon diameter and morphotype are not coupled, osteon diameters seem to be associated with increased strain magnitudes in some cases, but this is inconsistent. By contrast, osteon morphotypes are more strongly correlated with the distribution of tension and compression.
The crystallographic structure of Panicum Mosaic Virus (PMV).
Venue: Journal of structural biology

The structure of Panicum Mosaic Virus (PMV) was determined by X-ray diffraction analysis to 2.9Å resolution. The crystals were of pseudo symmetry F23; the true crystallographic unit cell was of space group P2(1) with a=411.7Å, b=403.9Å and c=412.5Å, with β=89.7°. The asymmetric unit was two entire T=3 virus particles, or 360 protein subunits. The structure was solved by conventional molecular replacement from two distant homologues, Cocksfoot Mottle Virus (CfMV) and Tobacco Necrosis Virus (TNV), of ∼20% sequence identity followed by phase extension. The model was initially refined with exact icosahedral constraints and then with icosahedral restraints. The virus has Ca(++) ions octahedrally coordinated by six aspartic acid residues on quasi threefold axes, which is completely different than for either CfMV or TNV. Amino terminal residues 1-53, 1-49 and 1-21 of the A, B and C subunits, respectively, and the four C-terminal residues (239-242) are not visible in electron density maps. The additional ordered residues of the C chain form a prominent "arm" that intertwines with symmetry equivalent "arms" at icosahedral threefold axes, as was seen in both CfMV and TNV. A 17 nucleotide hairpin segment of genomic RNA is icosahedrally ordered and bound at 60 equivalent sites at quasi twofold A-B subunit interfaces at the interior surface of the capsid. This segment of RNA may serve as a conformational switch for coat protein subunits, as has been proposed for similar RNA segments in other viruses.
Structural characterization of a D-isomer specific 2-hydroxyacid dehydrogenase from Lactobacillus delbrueckii ssp. bulgaricus.
Venue: Journal of structural biology

Hydroxyacid dehydrogenases, responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids in lactic acid producing bacteria, have a range of biotechnology applications including antibiotic synthesis, flavor development in dairy products and the production of valuable synthons. The genome of Lactobacillus delbrueckii ssp. bulgaricus, a member of the heterogeneous group of lactic acid bacteria, encodes multiple hydroxyacid dehydrogenases whose structural and functional properties remain poorly characterized. Here, we report the apo and coenzyme NAD⁺ complexed crystal structures of the L. bulgaricusD-isomer specific 2-hydroxyacid dehydrogenase, D2-HDH. Comparison with closely related members of the NAD-dependent dehydrogenase family reveals that whilst the D2-HDH core fold is structurally conserved, the substrate-binding site has a number of non-canonical features that may influence substrate selection and thus dictate the physiological function of the enzyme.
The Augmented Lagrange Multiplier Method for Exact Recovery of Corrupted Low-Rank Matrices
Venue: ArXiv

This paper proposes scalable and fast algorithms for solving the Robust PCA problem, namely recovering a low-rank matrix with an unknown fraction of its entries being arbitrarily corrupted. This problem arises in many applications, such as image processing, web data ranking, and bioinformatic data analysis. It was recently shown that under surprisingly broad conditions, the Robust PCA problem can be exactly solved via convex optimization that minimizes a combination of the nuclear norm and the l-norm . In this paper, we apply the method of augmented Lagrange multipliers (ALM) to solve this convex program. As the objective function is non-smooth, we show how to extend the classical analysis of ALM to such new objective functions and prove the optimality of the proposed algorithms and characterize their convergence rate. Empirically, the proposed new algorithms can be more than five times faster than the previous state-of-the-art algorithms for Robust PCA, such as the accelerated proximal gradient (APG) algorithm. Moreover, the new algorithms achieve higher precision, yet being less storage/memory demanding. We also show that the ALM technique can be used to solve the (related but somewhat simpler) matrix completion problem and obtain rather promising results too. We further prove the necessary and sufficient condition for the inexact ALM to converge globally. Matlab code of all algorithms discussed are available at http://perception.csl.illinois.edu/matrix-rank/home.html
Tooth periodontal ligament: Direct 3D microCT visualization of the collagen network and how the network changes when the tooth is loaded.
Venue: Journal of structural biology

The periodontal ligament (PDL), a soft tissue connecting the tooth and the bone, is essential for tooth movement, bone remodeling and force dissipation. A collagenous network that connects the tooth root surface to the alveolar jaw bone is one of the major components of the PDL. The organization of the collagenous component and how it changes under load is still poorly understood. Here using a state-of-the-art custom-made loading apparatus and a humidified environment inside a microCT, we visualize the PDL collagenous network of a fresh rat molar in 3D at 1 μm voxel size without any fixation or contrasting agents. We demonstrate that the PDL collagen network is organized in sheets. The spaces between sheets vary thus creating dense and sparse networks. Upon vertical loading, the sheets in both networks are stretched into well aligned arrays. The sparse network is located mainly in areas which undergo compressive loading as the tooth moves towards the bone, whereas the dense network functions mostly in tension as the tooth moves further from the bone. This new visualization method can be used to study other non-mineralized or partially mineralized tissues, and in particular those that are subjected to mechanical loads. The method will also be valuable for characterizing diseased tissues, as well as better understanding the phenotypic expressions of genetic mutants.
Glycerol inhibits water permeation through Plasmodium falciparum aquaglyceroporin.
Venue: Journal of structural biology

Plasmodium falciparum aquaglyceroporin (PfAQP) is a multifunctional membrane protein in the plasma membrane of P. falciparum, the parasite that causes the most severe form of malaria. The current literature has established the science of PfAQP's structure, functions, and hydrogen-bonding interactions but left unanswered the following fundamental question: does glycerol modulate water permeation through aquaglyceroporin that conducts both glycerol and water? This paper provides an affirmative answer to this question of essential importance to the protein's functions. On the basis of the chemical-potential profile of glycerol from the extracellular bulk region, throughout PfAQP's conducting channel, to the cytoplasmic bulk region, this study shows the existence of a bound state of glycerol inside aquaglyceroporin's permeation pore, from which the dissociation constant is approximately 14μM. A glycerol molecule occupying the bound state occludes the conducting pore through which permeating molecules line up in single file by hydrogen-bonding with one another and with the luminal residues of aquaglyceroporin. In this way, glycerol inhibits permeation of water and other permeants through aquaglyceroporin. The biological implications of this theory are discussed and shown to agree with the existent in vitro data. It turns out that the structure of aquaglyceroporin is perfect for the van der Waals interactions between the protein and glycerol to cause the existence of the bound state deep inside the conducting pore and, thus to play an unexpected but significant role in aquaglyceroporin's functions.
Crystal structure of the dimeric coiled-coil domain of the cytosolic nucleic acid sensor LRRFIP1.
Venue: Journal of structural biology

LRRFIP1 binds cytoplasmic double-stranded DNA and RNA and interacts with FLI, the mammalian homolog of Drosophila flightless I, through a highly conserved 87-amino acid domain. Upon binding nucleic acid ligands, LRRFIP1 recruits and activates β-catenin, leading to the IRF3-dependent production of type I interferon. However, the molecular mechanism of LRRFIP1 signaling is not well understood. Here we show that the FLI-interacting domain of LRRFIP1 forms a classic parallel, homodimeric coiled coil with 10 heptad repeats and 22 helical turns. The coiled coil domain is also a dimer in solution. However, a longer LRRFIP1 construct spanning the coiled coil and DNA binding domains assembles into higher order oligomers in solution. The structure of LRRFIP1-CC constitutes a valuable tool for probing the mechanism of LRRFIP1 signaling and for structural studies of larger LRRFIP1 constructs.
A ridge-based framework for segmentation of 3D electron microscopy datasets.
Venue: Journal of structural biology

Three-dimensional (3D) electron microscopy (EM) has become a major player in structural cell biology as it enables the analysis of subcellular architecture at an unprecedented level of detail. Interpretation of the resulting 3D volumes strongly depends on segmentation, which consists in decomposing the volume into their structural components. The computational approaches proposed so far have not turned out to be of general applicability. Thus, manual segmentation still remains a prevalent method. Here, a new computational framework for segmentation of 3D EM datasets is introduced. It relies on detection and characterization of ridges (i.e. local maxima). The detected ridges are modelled as asymmetric Gaussian functions whose parameters constitute ridge descriptors. This local information is then used to cluster the ridges, which leads to the ultimate segmentation. In this work we focus on membranes and locally planar structures in general. The performance of the framework is illustrated with its application to a number of complex 3D datasets and a quantitative analysis.
Vitrification of thick samples for soft X-ray cryo-tomography by high pressure freezing.
Venue: Journal of structural biology

Soft X-ray cryo-microscopy (cryo-XT) offers an ideal complement to electron cryo-microscopy (cryo-EM). Cryo-XT is applicable to samples more than an order of magnitude thicker than cryo-EM, albeit at a more modest resolution of tens of nanometers. Furthermore, the natural contrast obtained in the "water-window" by differential absorption by organic matter vs water yields detailed images of organelles, membranes, protein complexes, and other cellular components. Cryo-XT is thus ideally suited for tomography of eukaryotic cells. The increase in sample thickness places more stringent demands on sample preparation, however. The standard method for cryo-EM, i.e., plunging to a cryogenic fluid such as liquid ethane, is no longer ideally suited to obtain vitrification of thick samples for cryo-XT. High pressure freezing is an alternative approach, most closely associated with freeze-substitution and embedding, or with electron cryo-microscopy of vitreous sections (CEMOVIS). We show here that high pressure freezing can be adapted to soft X-ray tomography of whole vitrified samples, yielding a highly reliable method that avoids crystallization artifacts and potentially offers improved imaging conditions in samples not amenable to plunge-freezing.