nils-herrmann/clean_scirepeval huggingface dataset:
Enhancement of tomogram interpretability using the locked self-rotation function.
Venue: Journal of structural biology

The interpretation of cryo-electron tomograms of macromolecular complexes can be difficult because of the large amount of noise and because of the missing wedge effect. Here it is shown how the presence of rotational symmetry in a sample can be utilized to enhance the quality of a tomographic analysis. The orientation of symmetry axes in a sub-tomogram can be determined using a locked self-rotation function. Given this knowledge, the sub-tomogram density can then be averaged to improve its interpretability. Sub-tomograms of the icosahedral bacteriophage phiX174 are used to demonstrate the procedure.
Structural and functional insights into the role of a cupin superfamily isomerase in the biosynthesis of Choi moiety of aeruginosin.
Venue: Journal of structural biology

The 2-carboxy-6-hydroxyoctahydroindole (Choi) moiety is a hallmark of aeruginosins, a class of cyanobacterial derived bioactive linear tetrapeptides that possess antithrombotic activity. The biosynthetic pathway of Choi has yet to be resolved. AerE is a cupin superfamily enzyme that was shown to be involved in the biosynthesis of Choi, but its exact role remains unclear. This study reports the functional characterization and structural analyses of AerE. Enzymatic observation reveals that AerE can dramatically accelerate 1,3-allylic isomerization of the non-aromatic decarboxylation product of prephenate, dihydro-4-hydroxyphenylpyruvate (H2HPP). This olefin isomerization reaction can occur non-enzymatically and is the second step of the biosynthetic pathway from prephenate to Choi. The results of comparative structural analysis and substrate analogue binding geometry analysis combined with the results of mutational studies suggest that AerE employs an induced fit strategy to bind and stabilize the substrate in a particular conformation that is possibly favorable for 1,3-allylic isomerization of H2HPP through coordinate bonds, hydrogen bonds, π-π conjugation interaction and hydrophobic interactions. All of these interactions are critical for the catalytic efficiency.
Consideration of sample motion in cryo-tomography based on alignment residual interpolation.
Venue: Journal of structural biology

Recently, it has been shown that the resolution in cryo-tomography could be improved by considering the sample motion in tilt-series alignment and reconstruction, where a set of quadratic polynomials were used to model this motion. One requirement of this polynomial method is the optimization of a large number of parameters, which may limit its practical applicability. In this work, we propose an alternative method for modeling the sample motion. Starting from the standard fiducial-based tilt-series alignment, the method uses the alignment residual as local estimates of the sample motion at the 3D fiducial positions. Then, a scattered data interpolation technique characterized by its smoothness and a closed-form solution is applied to model the sample motion. The motion model is then integrated in the tomographic reconstruction. The new method improves the tomogram quality similar to the polynomial one, with the important advantage that the determination of the motion model is greatly simplified, thereby overcoming one of the major limitations of the polynomial model. Therefore, the new method is expected to make the beam-induced motion correction methodology more accessible to the cryoET community.
Breaking the long-standing morphological paradigm: Individual prisms in the pearl oyster shell grow perpendicular to the c-axis of calcite.
Venue: Journal of structural biology

Cross-sections of calcitic prismatic layers in mollusk shells, cut perpendicular to growth direction, reveal well-defined polygonal shapes of individual "grains" clearly visible by light and electron microscopy. For several kinds of shells, it was shown that the average number of edges in an individual prism approaches six during the growth process. Taking into account the rhombohedral symmetry of calcite, often presented in hexagonal axes, all this led to the long-standing opinion that calcitic prisms grow along the c-axis of calcite. In this paper, using X-ray diffraction and electron backscatter diffraction (EBSD), we unambiguously show that calcitic prisms in pearl oyster Pinctada margaritifera predominantly grow perpendicular to the c-axis. The obtained results imply that the hexagon-like habitus of growing crystallites may be not necessarily connected to calcite crystallography and, therefore, other factors should be taken into consideration. We analyze this phenomenon by comparing the organic contents in Pinctada margaritifera and Pinna nobilis shells, the later revealing regular growth of calcitic prisms along the c-axis.
Structure and electrochemistry of proteins harboring iron-sulfur clusters of different nuclearities. Part IV. Canonical, non-canonical and hybrid iron-sulfur proteins.
Venue: Journal of structural biology

A plethora of proteins are able to express iron-sulfur clusters, but have a clear picture of the different types of proteins and the different iron-sulfur clusters they harbor it is not easy. In the last five years we have reviewed structure/electrochemistry of metalloproteins expressing: (i) 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](Cys)3(nonthiolate ligand)} cores); (ii) metalloproteins harboring iron-sulfur centres of different nuclearities (namely: [4Fe-4S] and [2Fe-2S], [4Fe-4S] and [3Fe-4S], and [4Fe-4S], [3Fe-4S] and [2Fe-2S] clusters. Our target is now to review structure and electrochemistry of proteins harboring canonical, non-canonical and hybrid iron-sulfur proteins.
3D reconstruction of Trypanosoma cruzi-macrophage interaction shows the recruitment of host cell organelles towards parasitophorous vacuoles during its biogenesis.
Venue: Journal of structural biology

Trypanosoma cruzi has a complex life cycle where two infective developmental stages, known as trypomastigote and amastigote, can be found in the vertebrate host. Both forms can invade a large variety of cellular types and induce the formation of a parasitophorous vacuole (PV), that, posteriorly, disassembles and releases the parasites into the host cell cytoplasm. The biogenesis of T. cruzi PVs has not been analyzed in professional phagocytic cells. We investigated the biogenesis of PVs containing trypomastigotes or amastigotes in peritoneal macrophages. We observed the presence of profiles of the endoplasmic reticulum and lysosomes from the host cell near PVs at early stages of interaction in both developmental stages, suggesting that both organelles may participate as possible membrane donors for the formation of the PVs. The Golgi complex, however, was observed only near already formed PVs. Electron microscopy tomography and FIB-SEM microscopy followed by 3D reconstruction of entire PVs containing amastigotes or trypomastigotes confirmed the presence of both endoplasmic reticulum and lysosomes in the initial stages of PV formation. In addition, Golgi complex and mitochondria localize around PVs during their biogenesis. Taken together these observations provide a whole view of the invasion process in a professional phagocytic cell.
NADPH-dependent sulfite reductase flavoprotein adopts an extended conformation unique to this diflavin reductase.
Venue: Journal of structural biology

This is the first X-ray crystal structure of the monomeric form of sulfite reductase (SiR) flavoprotein (SiRFP-60) that shows the relationship between its major domains in an extended position not seen before in any homologous diflavin reductases. Small angle neutron scattering confirms this novel domain orientation also occurs in solution. Activity measurements of SiR and SiRFP variants allow us to propose a novel mechanism for electron transfer from the SiRFP reductase subunit to its oxidase metalloenzyme partner that, together, make up the SiR holoenzyme. Specifically, we propose that SiR performs its 6-electron reduction via intramolecular or intermolecular electron transfer. Our model explains both the significance of the stoichiometric mismatch between reductase and oxidase subunits in the holoenzyme and how SiR can handle such a large volume electron reduction reaction that is at the heart of the sulfur bio-geo cycle.
The FKBP12 subunit modifies the long-range allosterism of the ryanodine receptor.
Venue: Journal of structural biology

Ryanodine receptors (RyRs) are large conductance intracellular channels controlling intracellular calcium homeostasis in myocytes, neurons, and other cell types. Loss of RyR's constitutive cytoplasmic partner FKBP results in channel sensitization, dominant subconductance states, and increased cytoplasmic Ca2+. FKBP12 binds to RyR1's cytoplasmic assembly 130 Å away from the ion gate at four equivalent sites in the RyR1 tetramer. To understand how FKBP12 binding alters RyR1's channel properties, we studied the 3D structure of RyR1 alone in the closed conformation in the context of the open and closed conformations of FKBP12-bound RyR1. We analyzed the metrics of conformational changes of existing structures, the structure of the ion gate, and carried out multivariate statistical analysis of thousands of individual cryoEM RyR1 particles. We find that under closed state conditions, in the presence of FKBP12, the cytoplasmic domain of RyR1 adopts an upward conformation, whereas absence of FKBP12 results in a relaxed conformation, while the ion gate remains closed. The relaxed conformation is intermediate between the RyR1-FKBP12 complex closed (upward) and open (downward) conformations. The closed-relaxed conformation of RyR1 appears to be consistent with a lower energy barrier separating the closed and open states of RyR1-FKBP12, and suggests that FKBP12 plays an important role by restricting conformations within RyR1's conformational landscape.
Response of the tooth-periodontal ligament-bone complex to load: A microCT study of the minipig molar.
Venue: Journal of structural biology

One strategy evolved by teeth to avoid irreversible damage is to move and deform under the loads incurred during mastication. A key component in this regard is the periodontal ligament (PDL). The role of the bone underlying the PDL is less well defined. We study the interplay between the PDL and the underlying alveolar bone when loaded in the minipig. Using an Instron loading device we confirmed that the force-displacement curves of the molars and premolars of relatively fresh minipig intact mandibles are similar to those obtained for humans and other animals. We then used this information to obtain 3D images of the teeth before and after loading the tooth in a microCT such that the load applied is in the third linear part of the force displacement curve. We observed that at many locations there is a complimentary topography of the cementum and alveolar bone surface, strongly suggesting an active interplay between the tooth and the bone during mastication. We also observed that the loaded tooth does not come into direct contact with the underlying bone surface. A highly compressed layer of PDL is present between the tooth and the bone. The structure of the bone in the upper furcation region has a unique appearance with little obvious microstructure, abundant pores that have a large size range and at many locations the bone at the PDL interface has a needle-like shape. We conclude that there is a close interaction between the tooth, the PDL and the underlying alveolar bone during mastication. The highly compressed PDL layer that separates the tooth from the bone may fulfill a key shock absorbing function.
Rapid tilt-series acquisition for electron cryotomography.
Venue: Journal of structural biology

Using a new Titan Krios stage equipped with a single-axis holder, we developed two methods to accelerate the collection of tilt-series. We demonstrate a continuous-tilting method that can record a tilt-series in seconds, but with loss of details finer than ∼4 nm. We also demonstrate a fast-incremental method that can record a tilt-series several-fold faster than current methods and with similar resolution. We characterize the utility of both methods in real biological electron cryotomography workflows. We identify opportunities for further improvements in hardware and software and speculate on the impact such advances could have on structural biology.
Using neutron crystallography to elucidate the basis of selective inhibition of carbonic anhydrase by saccharin and a derivative.
Venue: Journal of structural biology

Up-regulation of carbonic anhydrase IX (CA IX) expression is an indicator of metastasis and associated with poor cancer patient prognosis. CA IX has emerged as a cancer drug target but development of isoform-specific inhibitors is challenging due to other highly conserved CA isoforms. In this study, a CA IXmimic construct was used (CA II with seven point mutations introduced, to mimic CA IX active site) while maintaining CA II solubility that make it amenable to crystallography. The structures of CA IXmimic unbound and in complex with saccharin (SAC) and a saccharin-glucose conjugate (SGC) were determined using joint X-ray and neutron protein crystallography. Previously, SAC and SGC have been shown to display CA isoform inhibitor selectivity in assays and X-ray crystal structures failed to reveal the basis of this selectivity. Joint X-ray and neutron crystallographic studies have shown active site residues, solvent, and H-bonding re-organization upon SAC and SGC binding. These observations highlighted the importance of residues 67 (Asn in CA II, Gln in CA IX) and 130 (Asp in CA II, Arg in CA IX) in selective CA inhibitor targeting.
Structural transformation-mediated dimerization of caspase recruitment domain revealed by the crystal structure of CARD-only protein in frog virus 3.
Venue: Journal of structural biology

Caspase recruitment domain (CARD)-only proteins (COPs), regulate apoptosis, inflammation, and innate immunity. They inhibit the assembly of NOD-like receptor complexes such as the inflammasome and NODosome, which are molecular complexes critical for caspase-1 activation. COPs are known to interact with either caspase-1 CARD or RIP2 CARD via a CARD-CARD interaction, and inhibit caspase-1 activation or further downstream signaling. In addition to the human COPs, Pseudo-ICE, INCA, and ICEBERG, several viruses also contain viral COPs that help them escape the host immune system. To elucidate the molecular mechanism of host immunity inhibition by viral COPs, we solved the structure of a viral COP for the first time. Our structure showed that viral COP forms a structural transformation-mediated dimer, which is unique and has not been reported in any structural study of a CARD domain. Based on the current structure, and the previously solved structures of other death domain superfamily members, we propose that structural transformation-mediated dimerization might be a new strategy for dimer assembly in the death domain superfamily.
Effects of the cardiomyopathy-causing E244D mutation of troponin T on the structures of cardiac thin filaments studied by small-angle X-ray scattering.
Venue: Journal of structural biology

Small-angle X-ray scattering experiments were carried out to investigate the structural changes of cardiac thin filaments induced by the cardiomyopathy-causing E244D mutation in troponin T (TnT). We examined native thin filaments (NTF) from a bovine heart, reconstituted thin filaments containing human cardiac wild-type Tn (WTF), and filaments containing the E244D mutant of Tn (DTF), in the absence and presence of Ca2+. Analysis by model calculation showed that upon Ca2+-activation, tropomyosin (Tm) and Tn in the WTF and NTF moved together in a direction to expose myosin-binding sites on actin. On the other hand, Tm and Tn of the DTF moved in the opposite directions to each other upon Ca2+-activation. These movements caused Tm to expose more myosin-binding sites on actin than the WTF, suggesting that the affinity of myosin for actin is higher for the DTF. Thus, the mutation-induced structural changes in thin filaments would increase the number of myosin molecules bound to actin compared with the WTF, resulting in the force enhancement observed for the E244D mutation.
Structural and functional characterization of TgpA, a critical protein for the viability of Pseudomonas aeruginosa.
Venue: Journal of structural biology

Pseudomonas aeruginosa is an opportunistic pathogen associated with severe diseases, such as cystic fibrosis. During an extensive search for novel essential genes, we identified tgpA (locus PA2873) in P. aeruginosa PAO1, as a gene playing a critical role in bacterial viability. TgpA, the translated protein, is an internal membrane protein with a periplasmic soluble domain, predicted to be endowed with a transglutaminase-like fold, hosting the Cys404, His448, and Asp464 triad. We report here that Cys404 mutation hampers the essential role of TgpA in granting P. aeruginosa viability. Moreover, we present the crystal structure of the TgpA periplasmic domain at 1.6 Å resolution as a first step towards structure-activity analysis of a new potential target for the discovery of antibacterial compounds.
Bending and branching of calcite laths in the foliated microstructure of pectinoidean bivalves occurs at coherent crystal lattice orientation.
Venue: Journal of structural biology

Foliated calcite is widely employed by some important pteriomorph bivalve groups as a construction material. It is made from calcite laths, which are inclined at a low angle to the internal shell surface, although their arrangement is different among the different groups. They are strictly ordered into folia in the anomiids, fully independent in scallops, and display an intermediate arrangement in oysters. Pectinids have particularly narrow laths characterized by their ability to change their growth direction by bending or winding, as well as to bifurcate and polyfurcate. Electron backscatter analysis indicates that the c-axes of laths are at a high, though variable, angle to the growth direction, and that the laths grow preferentially along the projection of an intermediate axis between two a-axes, although they can grow in any intermediate direction. Their main surfaces are not particular crystallographic faces. Analyses done directly on the lath surfaces demonstrate that, during the bending/branching events, all crystallographic axes remain invariant. The growth flexibility of pectinid laths makes them an excellent space-filling material, well suited to level off small irregularities of the shell growth surface. We hypothesize that the exceptional ability of laths to change their direction may be promoted by the mode of growth of biogenic calcite, from a precursor liquid phase induced by organic molecules.
Asymmetric protein design from conserved supersecondary structures.
Venue: Journal of structural biology

Computational design with supersecondary structures as building blocks has proven effective in the construction of new proteins with controlled geometries. So far, this approach has primarily exploited amplification, effectively harnessing the internal folding propensity of self-compatible fragments to achieve sufficient enthalpy for folding. Here we exploit an interface-driven strategy to depart from the repeat design realm, constructing an asymmetric, globular domain from heterologous supersecondary structures. We report the successful design of a dRP lyase domain fold, which agrees with the experimental NMR structure at atomic accuracy (backbone RMSD of 0.94 Å). Our results show that the residual folding information within conserved fragments, combined with efficient interface-directed sampling, can effectively yield globular proteins with novel sequences and biophysical properties.
Structural and functional characterization of a family GH53 β-1,4-galactanase from Bacteroides thetaiotaomicron that facilitates degradation of prebiotic galactooligosaccharides.
Venue: Journal of structural biology

Galactooligosaccharides (GOS) are prebiotic compounds synthesized from lactose using bacterial enzymes and are known to stimulate growth of beneficial bifidobacteria in the human colon. Bacteroides thetaiotaomicron is a prominent human colon commensal bacterial species that hydrolyzes GOS using an extracellular Glycosyl Hydrolase (GH) family GH53 endo-galactanase enzyme (BTGH53), releasing galactose-based products for growth. Here we dissect the molecular basis for GOS activity of this B. thetaiotaomicron GH53 endo-galactanase. Elucidation of its X-ray crystal structure revealed that BTGH53 has a relatively open active site cleft which was not observed with the bacterial enzyme from Bacillus licheniformis (BLGAL). BTGH53 acted on GOS with degree of polymerization ≤3 and therefore more closely resembles activity of fungal GH53 enzymes (e.g. Aspergillus aculeatus AAGAL and Meripileus giganteus MGGAL). Probiotic lactobacilli that lack galactan utilization systems constitute a group of bacteria with relevance for a healthy (infant) gut. The strains tested were unable to use GOS ≥ DP3. However, they completely consumed GOS in the presence of BTGH53, resulting in clear stimulation of their extent of growth. The extracellular BTGH53 enzyme thus may play an important role in carbohydrate metabolism in complex microbial environments such as the human colon. It also may find application for the development of synergistic synbiotics.
Structural insights into thermostabilization of leucine dehydrogenase from its atomic structure by cryo-electron microscopy.
Venue: Journal of structural biology

Leucine dehydrogenase (LDH, EC 1.4.1.9) is a NAD+-dependent oxidoreductase that catalyzes the deamination of branched-chain l-amino acids (BCAAs). LDH of Geobacillus stearothermophilus (GstLDH) is a highly thermostable enzyme that has been applied for the quantification or production of BCAAs. Here the cryo-electron microscopy (cryo-EM) structures of apo and NAD+-bound LDH are reported at 3.0 and 3.2 Å resolution, respectively. On comparing the structures, the two overall structures are almost identical, but it was observed that the partial conformational change was triggered by the interaction between Ser147 and the nicotinamide moiety of NAD+. NAD+ binding also enhanced the strength of oligomerization interfaces formed by the core domains. Such additional interdomain interaction is in good agreement with our experimental results showing that the residual activity of NAD+-bound form was approximately three times higher than that of the apo form after incubation at 80 °C. In addition, sequence comparison of three structurally known LDHs indicated a set of candidates for site-directed mutagenesis to improve thermostability. Subsequent mutation analysis actually revealed that non-conserved residues, including Ala94, Tyr127, and the C-terminal region, are crucial for oligomeric thermostability.
Metal centers in biomolecular solid-state NMR.
Venue: Journal of structural biology

Solid state NMR (SSNMR) has earned a substantial success in the characterization of paramagnetic systems over the last decades. Nowadays, the resolution and sensitivity of solid state NMR in biological molecules has improved significantly and these advancements can be translated into the study of paramagnetic biomolecules. However, the electronic properties of different metal centers affect the quality of their SSNMR spectra differently, and not all systems turn out to be equally easy to approach by this technique. In this review we will try to give an overview of the properties of different paramagnetic centers and how they can be used to increase the chances of experimental success.