nils-herrmann/clean_scirepeval huggingface dataset:
AgarFix: Simple and accessible stabilization of challenging single-particle cryo-EM specimens through crosslinking in a matrix of agar.
Venue: Journal of structural biology

Cryogenic electron microscopy (cryo-EM) allows structure determination of macromolecular assemblies that have resisted other structural biology approaches because of their size and heterogeneity. These challenging multi-protein targets are typically susceptible to dissociation and/or denaturation upon cryo-EM grid preparation, and often require crosslinking prior to freezing. Several approaches for gentle on-column or in-tube crosslinking have been developed. On-column crosslinking is not widely applicable because of the poor separation properties of gel filtration techniques. In-tube crosslinking frequently causes sample aggregation and/or precipitation. Gradient-based crosslinking through the GraFix method is more robust, but very time-consuming and necessitates specialised expensive equipment. Furthermore, removal of the glycerol typically involves significant sample loss and may cause destabilization detrimental to the sample quality. Here, we introduce an alternative procedure: AgarFix (Agarose Fixation). The sample is embedded in an agarose matrix that keeps the molecules separated, thus preventing formation of aggregates upon cross-inking. Gentle crosslinking is accomplished by diffusion of the cross-linker into the agarose drop. The sample is recovered by diffusion or electroelution and can readily be used for cryo-EM specimen preparation. AgarFix requires minimal equipment and basic lab experience, making it widely accessible to the cryo-EM community.
Exploring The Mechanism Of PPARγ Phosphorylation Mediated By CDK5.
Venue: Journal of structural biology

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor with a key role in metabolic processes and is target of CDK5 kinase phosphorylation at S245 (S273 in PPARγ isoform 2), thereby inducing insulin resistance. A remarkable effort has been addressed to find PPARγ ligands that inhibit S245 phosphorylation, but the poor understanding in this field challenges the design of such ligands. Here, through computational and biophysical methods, we explored an experimentally validated model of PPARγ-CDK5 complex, and we presented K261, K263 or K265, which are conserved in mammals, as important anchor residues for this interaction. In addition, we observed, from structural data analysis, that PPARγ ligands that inhibit S245 phosphorylation are not in direct contact with these residues; but induce structural modifications in PPARγ:CDK5/p25 interface. In summary, our PPARγ and CDK5/p25 interaction analyses open new possibilities for the rational design of novel inhibitors that impair S245 phosphorylation.
Solution structural model of the complex of the binding regions of human plasminogen with its M-protein receptor from Streptococcus pyogenes.
Venue: Journal of structural biology

VEK50 is a truncated peptide from a Streptococcal pyogenes surface human plasminogen (hPg) binding M-protein (PAM). VEK50 contains the full A-domain of PAM, which is responsible for its low nanomolar binding to hPg. The interaction of VEK50 with kringle 2, the PAM-binding domain in hPg (K2hPg), has been studied by high-resolution NMR spectroscopy. The data show that each VEK50 monomer in solution contains two tight binding sites for K2hPg, one each in the a1- (RH1; R17H18) and a2- (RH2; R30H31) repeats within the A-domain of VEK50. Two mutant forms of VEK50, viz., VEK50[RH1/AA] (VEK50ΔRH1) and VEK50[RH2/AA] (VEK50ΔRH2), were designed by replacing each RH with AA, thus eliminating one of the K2hPg binding sites within VEK50, and allowing separate study of each binding site. Using 13C- and 15N-labeled peptides, NMR-derived solution structures of VEK50 in its complex with K2hPg were solved. We conclude that the A-domain of PAM can accommodate two molecules of K2hPg docked within a short distance of each other, and the strength of the binding is slightly different for each site. The solution structure of the VEK50/K2hPg, complex, which is a reductionist model of the PAM/hPg complex, provides insights for the binding mechanism of PAM to a host protein, a process that is critical to S. pyogenes virulence.
The archaeal LDH-like malate dehydrogenase from Ignicoccus islandicus displays dual substrate recognition, hidden allostery and a non-canonical tetrameric oligomeric organization.
Venue: Journal of structural biology

The NAD(P)-dependent malate dehydrogenases (MalDHs) and NAD-dependent lactate dehydrogenases (LDHs) are homologous enzymes involved in central metabolism. They display a common protein fold and the same catalytic mechanism, yet have a stringent capacity to discriminate between their respective substrates. The MalDH/LDH superfamily is divided into several phylogenetically related groups. It has been shown that the canonical LDHs and LDH-like group of MalDHs are primarily tetrameric enzymes that diverged from a common ancestor. In order to gain understanding of the evolutionary history of the LDHs and MalDHs, the biochemical properties and crystallographic structure of the LDH-like MalDH from the hyperthermophilic archaeon Ignicoccus islandicus (I. isl) were determined. I. isl MalDH recognizes oxaloacetate as main substrate, but it is also able to use pyruvate. Surprisingly, with pyruvate, the enzymatic activity profile looks like that of allosteric LDHs, suggesting a hidden allosteric capacity in a MalDH. The I. isl MalDH tetrameric structure in the apo state is considerably different from those of canonical LDH-like MalDHs and LDHs, representing an alternative oligomeric organization. A comparison with MalDH and LDH counterparts provides strong evidence that the divergence between allosteric and non-allosteric members of the superfamily involves homologs with intermediate, atypical properties.
cryo_fit: Democratization of Flexible Fitting for Cryo-EM.
Venue: Journal of structural biology

Cryo-electron microscopy (cryo-EM) is becoming a method of choice for describing native conformations of biomolecular complexes at high resolution. The rapid growth of cryo-EM in recent years has created a high demand for automated solutions, both in hardware and software. Flexible fitting of atomic models to three-dimensional (3D) cryo-EM reconstructions by molecular dynamics (MD) simulation is a popular technique but often requires technical expertise in computer simulation. This work introduces cryo_fit, a package for the automatic flexible fitting of atomic models in cryo-EM maps using MD simulation. The package is integrated with the Phenix software suite. The module was designed to automate the multiple steps of MD simulation in a reproducible manner, as well as facilitate refinement and validation through Phenix. Through the use of cryo_fit, scientists with little experience in MD simulation can produce high quality atomic models automatically and better exploit the potential of cryo-EM.
Accuracy of MD solvent models in RNA structure refinement assessed via liquid-crystal NMR and spin relaxation data.
Venue: Journal of structural biology

Molecular dynamics (MD) simulations play an important role in characterizing Ribonucleic Acid (RNA) structure, augmenting information from experimental techniques such as Nuclear Magnetic Resonance (NMR). In this work, we examine the accuracy of structural representation resulting from application of a number of explicit and implicit solvent models and refinement protocols against experimental data ranging from high density of residual dipolar coupling (RDC) restraints to completely unrestrained simulations. For a prototype A-form RNA helix, our results indicate that AMBER RNA force field with either implicit or explicit solvent can produce a realistic dynamic representation of RNA helical structure, accurately cross-validating with respect to a diverse array of NMR observables. In refinement against NMR distance restraints, modern MD force fields are found to be equally adequate, with high fidelity cross-validation to the residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs), while slightly over-estimating structural order as monitored via NMR relaxation data. With restraints trimmed to encode only for base pairing information, cross-validation quality significantly deteriorates, now exhibiting a pronounced dependence on the choice of the solvent model. This deterioration is found to be partially reversible by increasing planarity restraints on the nucleobase geometry. For completely unrestrained MD simulations, the choice of water model becomes very important, with the best-performing TIP4P-Ew accurately reproducing both the RDC and RCSA data, while closely matching the NMR-derived order parameters. The information provided here will serve as a foundation for MD-based refinement of solution state NMR structures of RNA.
The peripheral vesicles gather multivesicular bodies with different behavior during the Giardia intestinalis life cycle.
Venue: Journal of structural biology

Giardia intestinalis presents an intriguing endomembrane system, which includes endoplasmic reticulum and peripheral vesicles (PVs). The PVs have previously been considered to be organelles that display early and late endosomal and lysosomal properties. Some of these vesicles accumulate macromolecules ingested by the protozoan and show acid phosphatase activity. It has been previously shown that the parasite releases microvesicles, which contribute to giardiasis pathogenesis; however, the vesicles' origin and the way in which they are released by the parasite still remain unclear. In this study, we induced the parasites to encyst in vitro and analyzed these events using advanced electron microscopy techniques, including focused ion beam and electron microscopy tomography followed by three-dimensional reconstruction, in order to better understand protozoal multivesicular body (MVB) biogenesis. In addition, we performed an ultrastructural analysis of phosphatase activity during differentiation. We demonstrated that some vegetative trophozoites' PVs exhibited morphological characteristics of MVBs with a mean diameter of 50 nm, containing intraluminal vesicles (ILVs).
Crystal structure of the CTLD7 domain of human M-type phospholipase A2 receptor.
Venue: Journal of structural biology

M-type phospholipase A2 receptor (PLA2R) is a member of the mannose receptor family. Recent evidence shows that PLA2R is a major autoantigen causing idiopathic membranous nephropathy (IMN), which is an autoimmune disease and one of the most common causes for nephrotic syndrome in adults. The epitope mapping data suggest that the major epitopes of PLA2R locate at the CysR, CTLD1 and CTLD7 domains. However, due to the lack of the high-resolution structural information, it is unclear how the autoantibodies interact with PLA2R. Here we determine the crystal structure of the CTLD7 domain of PLA2R at 1.8 Å, showing that it adopts a typical CTLD fold, and the structural alignments also provide hints for the potential antibody binding regions. In addition, the high-resolution structural information of CTLD7 could be applied to identify the epitopes for autoantibodies, which would facilitate the therapeutic strategies against IMN.
Structural basis for recognition and ring-cleavage of the Pseudomonas quinolone signal (PQS) by AqdC, a mycobacterial dioxygenase of the α/β-hydrolase fold family.
Venue: Journal of structural biology

The cofactor-less dioxygenase AqdC of Mycobacteroides abscessus catalyzes the cleavage and thus inactivation of the Pseudomonas quinolone signal (PQS, 2-heptyl-3-hydroxy-4(1H)-quinolone), which plays a central role in the regulation of virulence factor production by Pseudomonas aeruginosa. We present here the crystal structure of AqdC in its native state and in complex with the PQS cleavage product N-octanoylanthranilic acid, and of mutant AqdC proteins in complex with PQS. AqdC possesses an α/β-hydrolase fold core domain with additional helices forming a cap domain. The protein is traversed by a bipartite tunnel, with a funnel-like entry section leading to an elliptical substrate cavity where PQS positioning is mediated by a combination of hydrophobic interactions and hydrogen bonds, with the substrate's C4 carbonyl and C3 hydroxyl groups tethered by His97 and the catalytic His246, respectively. The side chain of the AqdC-bound product extends deeper into the "alkyl tail section" of the tunnel than PQS, tentatively suggesting product exit via this part of the tunnel. AqdC prefers PQS over congeners with shorter alkyl substituents at C2. Kinetic data confirmed the strict requirement of the active-site base His246 for catalysis, and suggested that evolution of the canonical nucleophile/His/Asp catalytic triad of the hydrolases to an Ala/His/Asp triad is favorable for catalyzing dioxygenolytic PQS ring cleavage.
An emergent understanding of strand displacement in RNA biology.
Venue: Journal of structural biology

DNA and RNA are generally regarded as central molecules in molecular biology. Recent advancements in the field of DNA/RNA nanotechnology successfully used DNA/RNA as programmable molecules to construct molecular machines and nanostructures with predefined shapes and functions. The key mechanism for dynamic control of the conformations of these DNA/RNA nanodevices is a reaction called strand displacement, in which one strand in a formed duplex is replaced by a third invading strand. While DNA/RNA strand displacement has mainly been used to de novo design molecular devices, we argue in this review that this reaction is also likely to play a key role in multiple cellular events such as gene recombination, CRISPR-based genome editing, and RNA cotranscriptional folding. We introduce the general mechanism of strand displacement reaction, give examples of its use in the construction of molecular machines, and finally review natural processes having characteristic which suggest that strand displacement is occurring.
A surrogate structural platform informed by ancestral reconstruction and resurrection of a putative carbohydrate binding module hybrid illuminates the neofunctionalization of a pectate lyase.
Venue: Journal of structural biology

Yersinia enterocolitica is a pectinolytic zoonotic foodborne pathogen, the genome of which contains pectin-binding proteins and several different classes of pectinases, including polysaccharide lyases (PLs) and an exopolygalacturonase. These proteins operate within a coordinated pathway to completely saccharify homogalacturonan (HG). Polysaccharide lyase family 2 (PL2) is divided into two major subfamilies that are broadly-associated with contrasting 'endolytic' (PL2A) or 'exolytic' (PL2B) activities on HG. In the Y. enterocolitica genome, the PL2A gene is adjacent to an independent carbohydrate binding module from family 32 (YeCBM32), which possesses a N-terminal secretion tag and is known to specifically bind HG. Independent CBMs are rare in nature and, most commonly, are fused to enzymes in order to potentiate catalysis. The unconventional gene architecture of YePL2A and YeCBM32, therefore, may represent an ancestral relic of a fission event that decoupled PL2A from its cognate CBM. To provide further insight into the evolution of this pectinolytic locus and the molecular basis of HG depolymerisation within Y. enterocolitica, we have resurrected a YePL2A-YeCBM32 chimera and demonstrated that the extant PL2A digests HG more efficiently. In addition, we have engineered a tryptophan from the active site of the exolytic YePL2B into YePL2A (YePL2A-K291W) and demonstrated, using X-ray crystallography of substrate complexes, that it is a structural determinant of exo-activity within the PL2 family. In this manner, surrogate structural platforms may assist in the study of phylogenetic relationships informed by extant and resurrected sequences, and can be used to overcome challenging structural problems within carbohydrate active enzyme families.
3.1 Å structure of yeast RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion solved using streptavidin affinity grids.
Venue: Journal of structural biology

Despite significant advances in all aspects of single particle cryo-electron microscopy (cryo-EM), specimen preparation still remains a challenge. During sample preparation, macromolecules interact with the air-water interface, which often leads to detrimental effects such as denaturation or adoption of preferred orientations, ultimately hindering structure determination. Randomly biotinylating the protein of interest (for example, at its primary amines) and then tethering it to a cryo-EM grid coated with two-dimensional crystals of streptavidin (acting as an affinity surface) can prevent the protein from interacting with the air-water interface. Recently, this approach was successfully used to solve a high-resolution structure of a test sample, a bacterial ribosome. However, whether this method can be used for samples where interaction with the air-water interface has been shown to be problematic remains to be determined. Here we report a 3.1 Å structure of an RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion (Pol II EC(CPD)) solved using streptavidin grids. Our previous attempt to solve this structure using conventional sample preparation methods resulted in a poor quality cryo-EM map due to Pol II EC(CPD)'s adopting a strong preferred orientation. Imaging the same sample on streptavidin grids improved the angular distribution of its view, resulting in a high-resolution structure. This work shows that streptavidin affinity grids can be used to address known challenges posed by the interaction with the air-water interface.
VfoldLA: A web server for loop assembly-based prediction of putative 3D RNA structures.
Venue: Journal of structural biology

RNA three-dimensional (3D) structures are critical for RNA cellular functions. However, structure prediction for large and complex RNAs remains a challenge, which hampers our understanding of RNA structure-function relationship. We here report a new web server, the VfoldLA server (http://rna.physics.missouri.edu/vfoldLA), for the prediction of RNA 3D structures from nucleotide sequences and base-pair information (2D structure). This server is based on the recently developed VfoldLA, a model that classifies the single-stranded loops (junctions) into four different types and according to the loop-helix connections, assembles RNA 3D structures from the loop/junction templates. The VfoldLA web server provides a user-friendly online interface for a fully automated prediction of putative 3D RNA structures using VfoldLA. With a single-RNA or RNA-RNA complex sequence and 2D structure as input, the server generates structure(s) with the JSmol visualization along with a downloadable PDB file. The output result may serve as useful scaffolds for future structure refinement studies.
Delving into the amyloidogenic core of human leukocyte chemotactic factor 2.
Venue: Journal of structural biology

ALECT2 (leukocyte chemotactic factor 2) amyloidosis is one of the most recently identified amyloid-related diseases, with LECT2 amyloids commonly found in different types of tissues. Under physiological conditions, LECT2 is a 16 kDa multifunctional protein produced by the hepatocytes and secreted into circulation. The pathological mechanisms causing LECT2 transition into the amyloid state are still largely unknown. In the case of ALECT2 patients, there is no disease-causing mutation, yet almost all patients carry a common polymorphism that appears to be necessary but not sufficient to directly trigger amyloidogenesis. In this work, we followed a reductionist methodology in order to detect critical amyloidogenic "hot-spots" during the fibrillation of LECT2. By associating experimental and computational assays, this approach reveals the explicit amyloidogenic core of human LECT2 and pinpoints regions with distinct amyloidogenic properties. The fibrillar architecture of LECT2 polymers, based on our results, provides a wealth of detailed information about the amyloidogenic "hot-spot" interactions and represents a starting point for future peptide-driven intervention in ALECT2 amyloidosis.
Resolving the conformational dynamics of ErbB growth factor receptor dimers.
Venue: Journal of structural biology

The combinatorial dimerization of the ErbB growth factor receptors (ErbB1- ErbB4) are critical for their function. Here, we have characterized the conformational dynamics of ErbB transmembrane homo-dimers and hetero-dimers by using a coarse-grain simulation framework. All dimers, except ErbB4-4 and ErbB1-4, exhibit at least two conformations. The reported NMR structures correspond to one of these conformations, representing the N-terminal active state in ErbB1-1 (RH2), ErbB2-2 (RH1) and ErbB4-4 (RH) homo-dimers and the LH dimer in ErbB3-3 homo-dimer, validating the computational approach. Further, we predict a right-handed ErbB3-3 dimer conformer that warrants experimental testing. The five hetero-dimers that have not yet been experimentally resolved display prominent right-handed dimers associating by the SmXXXSm motif. Our results provide insights into the constitutive signaling of ErbB4 after cleavage of the extracellular region. The presence of the inactive-like dimer conformers leading to symmetric kinase domains gives clues on the autoinhibition of the receptor dimers. The dimer states characterized here represent an important step towards understanding the combinatorial cross associations in the ErbB family.
Biomineralization of dentin.
Venue: Journal of structural biology

A single biomineralization of demineralized dentin is significant to restore the demineralized dentin due to dental caries or erosion. In recent years, meaningful progress has been made regarding the mechanisms involved in the biomineralization of dentin collagen. Concepts changing from the classical ion-based crystallization to non-classical particle-based crystallization, inspired a different strategy to infiltrate the demineralized dentin collagen. The remarkable discovery was the report of liquid-like amorphous calcium phosphate as nanoprecursor particles to carbonated hydroxyapatite. The non-collagenous proteins and their analogues are widely investigated, for their key role in controlling mineralization during the process of crystal nucleation and growth. The in-depth studies of the gap zone provided significant improvements in our understanding of the structure of collagen and of the intrafibrillar remineralization of collagen fibrils. The collagen is not a passive substrate as previously supposed, and the active role of guiding nanoprecursor infiltration and mediating its nucleation has been demonstrated. Furthermore, recovery of mechanical properties has been evaluated to determine the effectiveness of dentin remineralization. Finally, the problems regarding the origin formation of the calcium phosphate that is deposited in the collagen, and the exact interactions between the non-collagenous proteins, amorphous calcium phosphate and collagen are still unclear. We reviewed the importance of these findings in enriching our understanding of dentin biomineralization, while addressing certain limitations that are inherent to in vitro studies.