Structure Determination Research Articles

X-ray crystal structure of a designed rigidified imaging scaffold in the ligand-free conformation.

Imaging scaffolds composed of designed protein cages fused to designed ankyrin repeat proteins (DARPins) have enabled the structure determination of small proteins by cryogenic electron microscopy (cryo-EM). One particularly well characterized scaffold type is a symmetric tetrahedral assembly composed of 24 subunits, 12 A and 12 B, which has three cargo-binding DARPins positioned on each vertex. Here, the X-ray crystal structure of a representative tetrahedral scaffold in the apo state is reported at 3.8 Å resolution. The X-ray crystal structure complements recent cryo-EM findings on a closely related scaffold, while also suggesting potential utility for crystallographic investigations. As observed in this crystal structure, one of the three DARPins, which serve as modular adaptors for binding diverse `cargo' proteins, present on each of the vertices is oriented towards a large solvent channel. The crystal lattice is unusually porous, suggesting that it may be possible to soak crystals of the scaffold with small (≤30 kDa) protein cargo ligands and subsequently determine cage-cargo structures via X-ray crystallography. The results suggest the possibility that cryo-EM scaffolds may be repurposed for structure determination by X-ray crystallography, thus extending the utility of electron-microscopy scaffold designs for alternative structural biology applications.

Key Technology Research and Equipment Development of Automatic Spool Feeding Device

To solve the problems of high labour intensity and low efficiency of artificial spool feeding, an automatic spool feeding device is developed. SolidWorks is used for overall structural scheme design and determination of design parameters. The kinematic equation of the chain conveyor line is established, a mathematical model is solved with MATLAB, and sprocket, and chain parameters are selected. The clamping mechanism and turnover mechanism are designed, theoretical analysis of the key structure is completed. Dynamic simulation of the chain conveyor line is carried out by ADAMS, and preliminary operational stability is determined by combining motion curves. The test prototype is developed for feeding tests and measurement of the operating parameters. The test results show that the efficiency of machine feeding is 13% higher than that of manual feeding, the average feeding time is 43% lower, the halt waiting time is 43% lower, and the operating parameters are by the theoretical design and the work is reliable. The research results can provide a reference for similar spool-feeding devices.

DomainFit: Identification of protein domains in cryo-EM maps at intermediate resolution using AlphaFold2-predicted models.

Cryoelectron microscopy (cryo-EM) has revolutionized the structural determination of macromolecular complexes. With the paradigm shift to structure determination of highly complex endogenous macromolecular complexes exvivo and in situ structural biology, there are an increasing number of structures of native complexes. These complexes often contain unidentified proteins, related to different cellular states or processes. Identifying proteins at resolutions lower than 4Å remains challenging because side chains cannot be visualized reliably. Here, we present DomainFit, a program for semi-automated domain-level protein identification from cryo-EM maps, particularly at resolutions lower than 4Å. By fitting domains from AlphaFold2-predicted models into cryo-EM maps, the program performs statistical analyses and attempts to identify the domains and protein candidates forming the density. Using DomainFit, we identified two microtubule inner proteins, one of which contains a CCDC81 domain and is exclusively localized in the proximal region of the doublet microtubule in Tetrahymena thermophila.

Precise Structural Analysis of Neutral Glycans Using Aerolysin Mutant T240R Nanopore.

Glycans play vital roles in nearly all life processes of multicellular organisms, and understanding these activities is inseparable from elucidating the biological significance of glycans. However, glycan research has lagged behind that of DNA and protein due to the challenges posed by structural heterogeneity and isomerism (i.e., structures with equal molecular weights) the lack of high-efficiency structural analysis techniques. Nanopore technology has emerged as a sensitive single-molecule biosensor, shining a light on glycan analysis. However, a significant number of glycans are small and uncharged, making it challenging to elicit identifiable nanopore signals. Here we introduce a R-binaphthyl tag into glycans, which enhances the cation-π interaction between the derivatized glycan molecules and the nanopore interface, enabling the detection of neutral glycans with an aerolysin nanopore. This approach allows for the distinction of di-, tri-, and tetrasaccharides with monosaccharide resolution and has the potential for group discrimination, the monitoring of enzymatic transglycosylation reactions. Notably, the aerolysin mutant T240R achieves unambiguous identification of six disaccharide isomers, trisaccharide and tetrasaccharide linkage isomers. Molecular docking simulations reveal that multiple noncovalent interactions occur between residues R282, K238, and R240 and the glycans and R-binaphthyl tag, significantly slowing down their translocation across the nanopore. Importantly, we provide a demonstration of the kinetic translocation process of neutral glycan isomers, establishing a solid theoretical foundation for glycan nanopore analysis. The development of our technology could promote the analysis of glycan structural isomers and has the potential for nanopore-based glycan structural determination and sequencing.

Structural elucidation of the mesothelin-mucin-16/CA125 interaction.

Mesothelin (MSLN) is a cell-surface glycoprotein expressed at low levels on normal mesothelium but overexpressed in many cancers. Mesothelin has been implicated to play role/s in cell adhesion and multiple signaling pathways. Mucin-16/CA125 is an enormous cell-surface glycoprotein, also normally expressed on mesothelium and implicated in the progression and metastasis of several cancers, and directly binds mesothelin. However, the precise biological function/s of mesothelin and mucin-16/CA125 remain mysterious. We report protein engineering and recombinant production, qualitative and quantitative binding studies, and a crystal structure determination elucidating the molecular-level details governing recognition of mesothelin by mucin-16/CA125. The interface is small, consistent with the ∼micromolar binding constant and is free of glycan-mediated interactions. Sequence comparisons and modeling suggest that multiple mucin-16/CA125 modules can interact with mesothelin through comparable interactions, potentially generating a high degree of avidity at the cell surface to overcome the weak affinity, with implications for functioning and therapeutic interventions.

Disordered layers and dimerization in the crystal structure of TaOCl2

Understanding the properties of transition metal materials requires consideration of the competition between the various types of electrons that comprise the system, whether they be delocalized and metallic, localized and magnetic, or localized and bonded. Here we report the refined crystal structure determination and physical properties of the two-dimensional layered compound TaOCl2. Within each layer we find a strong in-plane dimerization of Ta atoms perpendicular to the Ta-O-Ta chains, with significant disorder in the positioning of dimers in adjacent layers, suggesting that the free electron of each Ta localizes in Ta-Ta bonds. This is supported by resistivity measurements (ρ300K = 4.4*108 Ω-cm) as well as featureless, temperature-independent magnetic susceptibility up to T = 750 K. Analysis of the phonon contribution to specific heat shows the presence of a localized Einstein mode with TE = 175 K, attributed to the localized vibrations of the Ta-Ta dimers. Density functional theory calculations show that the magnitude of the Ta displacements (0.24 Å) has a dramatic effect on the electronic structure, changing from a metal with a bandwidth of 3.2 eV to an insulator with a flat band just below EF. Our results show how direct exchange interactions between Ta atoms that are relatively far from each other can still play a central role in the behavior of 5d transition metal compounds, taking advantage of the extended 5d atomic orbitals, and produce localized dimers with a bond distance of approximately 2.84 Å.

Harnessing the 3D-Beacons Network: A Comprehensive Guide to Accessing and Displaying Protein Structure Data.

Recent advancements in protein structure determination and especially in protein structure prediction techniques have led to the availability of vast amounts of macromolecular structures. However, the accessibility and integration of these structures into scientific workflows are hindered by the lack of standardization among publicly available data resources. To address this issue, we introduced the 3D-Beacons Network, a unified platform that aims to establish a standardized framework for accessing and displaying protein structure data. In this article, we highlight the importance of standardized approaches for accessing protein structure data and showcase the capabilities of 3D-Beacons. We describe four protocols for finding and accessing macromolecular structures from various specialist data resources via 3D-Beacons. First, we describe three scenarios for programmatically accessing and retrieving data using the 3D-Beacons API. Next, we show how to perform sequence-based searches to find structures from model providers. Then, we demonstrate how to search for structures and fetch them directly into a workflow using JalView. Finally, we outline the process of facilitating access to data from providers interested in contributing their structures to the 3D-Beacons Network. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Programmatic access to the 3D-Beacons API Basic Protocol 2: Sequence-based search using the 3D-Beacons API Basic Protocol 3: Accessing macromolecules from 3D-Beacons with JalView Basic Protocol 4: Enhancing data accessibility through 3D-Beacons.

Polymorphic Structure Determination of the Macrocyclic Drug Paritaprevir by MicroED (Adv. Biology 5/2024)

Polymorphic Structure Determination of the Macrocyclic Drug Paritaprevir by MicroED (Adv. Biology 5/2024)

Collision-Induced Dissociation of Fucose and Identification of Anomericity.

Structural determination of carbohydrates using mass spectrometry remains challenging, particularly, the differentiation of anomeric configurations. In this work, we studied the collision-induced dissociation (CID) mechanisms of sodiated α- and β-l-fucose using an experimental method and quantum chemistry calculations. The calculations show that α-l-fucose is more likely to undergo dehydration due to the fact that O1 and O2 are on the same side of the sugar ring. In contrast, β-l-fucose is more prone to the ring-opening reaction because more OH groups are on the same side of the sugar ring as O1. These differences suggest a higher preference for the dehydration reaction in sodiated α-l-fucose but a lower preference for ring-opening compared to that of β-l-fucose. The calculation results, which are used to assign the CID mass spectra of α- and β-l-fucose separated by high-performance liquid chromatography, are supported by the fucose produced from the CID of disaccharides Fuc-β-(1 → 3)-GlcNAc and Fuc-α-(1 → 4)-GlcNAc. This study demonstrates that the correlation of cis- and trans-configurations of O1 and O2 to the relative branching ratios of dehydration and cross-ring dissociation in CID, observed in aldohexose and ketohexose in the pyranose form, can be extended to deoxyhexoses for anomericity determination.

1H, 15N and 13C resonance backbone and side-chain assignments and secondary structure determination of the BRCT domain of Mtb LigA.

The BRCA1 carboxyl-terminal (BRCT) domain, an evolutionarily conserved structural motif, is ubiquitous in a multitude of proteins spanning prokaryotic and eukaryotic organisms. In Mycobacterium tuberculosis (Mtb), BRCT domain plays a pivotal role in the catalytic activity of the NAD+-dependent DNA ligase (LigA). LigA is pivotal in DNA replication, catalyzing the formation of phosphodiester bonds in Okazaki fragments and repairing single-strand breaks in damaged DNA, essential for the survival of Mtb. Structural and functional aspects of LigA unveil its character as a highly modular protein, undergoing substantial conformational changes during its catalytic cycle. Although the BRCT domain of Mtb LigA plays an essential role in DNA binding and protein-protein interactions, the precise mechanism of action remains poorly understood. Unravelling the structure of the BRCT domain holds the promise of advancing our understanding of this pivotal domain. Additionally, it will facilitate further exploration of the protein-protein interactions and enhance our understanding of inter domain interactions within LigA, specifically between BRCT and the Adenylation domain. In this study, we demonstrate the overexpression of the BRCT domain of Mtb LigA and conduct its analysis using solution NMR spectroscopy, revealing a well-folded structure and we present the nearly complete chemical shift assignments of both backbone and sidechains. In addition, a secondary structure prediction by TALOS N predicts BRCT consisting of 3 α-helices and 4 β-sheets, closely resembling the typical structural topology of most BRCT domains.

Thiosalicylanilide complexes of nickel(II), palladium(II) and platinum(II)

Reactions of the complexes cis-[PtCl2(PPh3)2], [PtCl2(dppf)] {dppf = Fe(C5H4PPh2)2}, [PdCl2(PPh3)2], [PdCl2(dppe)] (dppe = Ph2PCH2CH2PPh2) and [NiCl2(dppe)] with thiosalicylanilide (HSC6H4CONHPh, H2-1a) and its substituted derivatives H2-1b to H2-1d (where H2-1b = p-Cl-HSC6H4CONHPh, H2-1c = p-CH3-HSC6H4CONHPh, and H2-1d = m-OCH3-HSC6H4CONHPh) gave a series of square-planar MS2P2-type complexes. The complexes were characterised by ESI MS, 1H and 31P{1H} NMR, FTIR, and microelemental analyses, and in some cases by single crystal X-ray crystallography. The predominant coordination mode is through bonding of deprotonated thiol group of two ligands. However, the tendency to form complexes with a single, chelating S,N- and S,O-donor ligand was observed by 31P{1H} NMR in some reactions. In addition, cis/trans isomerisation was observed in some complexes by 31P{1H} NMR and confirmed by X-ray structure determinations on trans-[Pt(H-1b)2(PPh3)2] and trans-[Pd(H-1b)2(PPh3)2]. A comparison of the energetics of the reaction systems involving [Pt(1b)(PPh3)2]/[Pt(H-1b)2(PPh3)2], [Pt(1d)(PPh3)2]/[Pt(H-1d)2(PPh3)2] and [Pd(1b)(dppe)]/[Pd(H-1b)2(dppe)] using Density Functional Theory (DFT) revealed that the MS2P2 systems [Pt(H-1b)2(PPh3)2], [Pt(H-1d)2(PPh3)2] and [Pd(H-1b)2(dppe)] have lower Gibbs free energies than the S,N-chelate systems [Pt(1b)(PPh3)2], [Pt(1d)(PPh3)2] and [Pd(1b)(dppe)], and are thermodynamically favoured. Intermolecular interactions quantified and visualized on Hirshfeld surfaces for trans-[Pt(H-1b)2(PPh3)2], [Pt(H-1d)2(PPh3)2].H2O, and [Pd(H-1a)2(dppe)] revealed the dominant contribution of H···H contacts to crystal packing.

Neutron diffraction: a primer

Abstract Because of the neutron’s special properties, neutron diffraction may be considered one of the most powerful techniques for structure determination of crystalline and related matter. Neutrons can be released from nuclear fission, from spallation processes, and also from low-energy nuclear reactions, and they can then be used in powder, time-of-flight, texture, single crystal, and other techniques, all of which are perfectly suited to clarify crystal and magnetic structures. With high neutron flux and sufficient brilliance, neutron diffraction also excels for diffuse scattering, for in situ and operando studies as well as for high-pressure experiments of today’s materials. For these, the wave-like neutron’s infinite advantage (isotope specific, magnetic) is crucial to answering important scientific questions, for example, on the structure and dynamics of light atoms in energy conversion and storage materials, magnetic matter, or protein structures. In this primer, we summarize the current state of neutron diffraction (and how it came to be), but also look at recent advances and new ideas, e.g., the design of new instruments, and what follows from that.

Further Insight into the Manganese(II) 2,2′-Bipyridine-1,1′-dioxide Homoleptic Complex: Single-Crystal X-ray Structure Determination of the Perchlorate Salt and DFT Calculations

The homoleptic cationic complex formed by reacting suitable manganese(II) salts with 2,2′-bipyridine-1,1′-dioxide (bipyO2) has been subjected to several studies in the past because of its peculiar absorption and electrochemical features. Here, the first single-crystal X-ray structure determination of a [Mn(bipyO2)3]2+ salt is reported, where the charge of the cation is balanced by perchlorate anions. The hydrated salt [Mn(bipyO2)3](ClO4)2 crystallizes in the monoclinic system (P21/n space group) and the asymmetric unit contains three cationic complexes and six perchlorate anions. The environment of the manganese(II) ions is best described as octahedral, with scarce variations among the three cations in the asymmetric unit. The bipyO2 ligands exhibit κ2 coordination mode, forming seven-membered metallacycles. The X-ray outcomes have been used as the starting point for DFT and TDDFT calculations, aimed to elucidate the charge transfer origin of the noticeable absorption in the visible range. The MLCT nature is confirmed by the hole and electron distributions associated with the spin-allowed transitions. DFT calculations on the related manganese(III) complex indicate that the geometry of [Mn(bipyO2)3]2+ changes only slightly upon oxidation, in agreement with the reversible electrochemical behaviour experimentally observed.

Structural basis of closed groove scrambling by a TMEM16 protein.

Activation of Ca2+-dependent TMEM16 scramblases induces phosphatidylserine externalization, a key step in multiple signaling processes. Current models suggest that the TMEM16s scramble lipids by deforming the membrane near a hydrophilic groove and that Ca2+ dependence arises from the different association of lipids with an open or closed groove. However, the molecular rearrangements underlying groove opening and how lipids reorganize outside the closed groove remain unknown. Here we directly visualize how lipids associate at the closed groove of Ca2+-bound fungal nhTMEM16 in nanodiscs using cryo-EM. Functional experiments pinpoint lipid-protein interaction sites critical for closed groove scrambling. Structural and functional analyses suggest groove opening entails the sequential appearance of two π-helical turns in the groove-lining TM6 helix and identify critical rearrangements. Finally, we show that the choice of scaffold protein and lipids affects the conformations of nhTMEM16 and their distribution, highlighting a key role of these factors in cryo-EM structure determination.

Molecular regulation of calcium-sensing receptor (CaSR)-mediated signaling.

Calcium-sensing receptor (CaSR), a family C G-protein-coupled receptor, plays a crucial role in regulating calcium homeostasis by sensing small concentration changes of extracellular Ca2+, Mg2+, amino acids (e.g., L-Trp and L-Phe), small peptides, anions (e.g., HCO3 - and PO4 3-), and pH. CaSR-mediated intracellular Ca2+ signaling regulates a diverse set of cellular processes including gene transcription, cell proliferation, differentiation, apoptosis, muscle contraction, and neuronal transmission. Dysfunction of CaSR with mutations results in diseases such as autosomal dominant hypocalcemia, familial hypocalciuric hypercalcemia, and neonatal severe hyperparathyroidism. CaSR also influences calciotropic disorders, such as osteoporosis, and noncalciotropic disorders, such as cancer, Alzheimer's disease, and pulmonary arterial hypertension. This study first reviews recent advances in biochemical and structural determination of the framework of CaSR and its interaction sites with natural ligands, as well as exogenous positive allosteric modulators and negative allosteric modulators. The establishment of the first CaSR protein-protein interactome network revealed 94 novel players involved in protein processing in endoplasmic reticulum, trafficking, cell surface expression, endocytosis, degradation, and signaling pathways. The roles of these proteins in Ca2+-dependent cellular physiological processes and in CaSR-dependent cellular signaling provide new insights into the molecular basis of diseases caused by CaSR mutations and dysregulated CaSR activity caused by its protein interactors and facilitate the design of therapeutic agents that target CaSR and other family C G-protein-coupled receptors.

Three-dimensional domain identification in a single hexagonal manganite nanocrystal

The three-dimensional domain structure of ferroelectric materials significantly influences their properties. The ferroelectric domain structure of improper multiferroics, such as YMnO3, is driven by a non-ferroelectric order parameter, leading to unique hexagonal vortex patterns and topologically protected domain walls. Characterizing the three-dimensional structure of these domains and domain walls has been elusive, however, due to a lack of suitable imaging techniques. Here, we present a multi-peak Bragg coherent x-ray diffraction imaging determination of the domain structure in single YMnO3 nanocrystals. We resolve two ferroelectric domains separated by a domain wall and confirm that the primary atomic displacements occur along the crystallographic c-axis. Correlation with atomistic simulations confirms the Mexican hat symmetry model of domain formation, identifying two domains with opposite ferroelectric polarization and adjacent trimerization, manifesting in a clockwise arrangement around the hat’s brim.

PELESTARIAN DAN PEMBERDAYAAN SUMBER DAYA ALAM DESA MULYASARI MELALUI KEGIATAN KULIAH KERJA NYATA UNSIKA

Mulyasari Village is one of the villages in Ciampel sub-district, Karawang Regency, West Java, Indonesia with a population of 6,817 in 2024. The village has a number of potentials that can be developed, especially in agriculture. However, this potential has not been optimized to the optimum. The Mulyasari Village community still has limitations in developing this potential, and there are still a number of problems that must be given solutions, including the lack of public awareness of waste management, stunting problems, rampant bullying cases, community businesses that have not kept up with the times, the absence of activities for 3 months at the green house in Mulyasari Village, and the absence of a reading corner at the Mulyasari Village preschool. In general, the goal of Singaperbangsa University (UNSIKA) Community Service Program (KKN) in the context of community service is to improve the standard of living of the community through a series of UNSIKA KKN programs and activities implemented by UNSIKA students. The method used in this KKN is an extension method consisting of six stages of activities, namely: 1) Determination of the organizational structure. 2) Observation of KKN partners. 3) Formulation of work programs by adjusting the observation results. 3) Implementation. 4) Evaluation of activities. 5) Final report. The UNSIKA KKN program in Mulyasari Village produced seven excellent programs to improve the level of health, education, economy, and environment of the community in Mulyasari Village. These programs include: 1) Stunting socialization with the provision of PMT. 2) Logo and Social Media creation for Soy Milk UMKM. 3) Literacy learning media for all PAUD in Mulyasari village. 4) Making road signs on several sides of the mulyasari village road. 5) Reviving the Hydroponic Green House. 6) Produce solid and liquid organic fertilizer composter. 7) Conducting anti-bullying and ethics socialization activities for all elementary schools in Mulyasari village. Based on the observations and analysis made by UNSIKA students during the KKN activities, it can be concluded that Mulyasari Village has great potential to be developed. With good and optimal management, these potentials can improve the welfare of the people of Mulyasari Village.

A Water-Soluble Wavy Coordination Polymer of Cu(II) as a Turn-On Luminescent Probe for Histidine and Histidine-Rich Proteins/Peptides.

Histidine plays an essential role in most biological systems. Changes in the homeostasis of histidine and histidine-rich proteins are connected to several diseases. Herein, we report a water-soluble Cu(II) coordination polymer, labeled CuCP, for the fluorimetric detection of histidine and histidine-rich proteins and peptides. Single-crystal structure determination of CuCP revealed a two-dimensional wavy network structure in which a carboxylate group connects the individual Cu(II) dimer unit in a syn-anti conformation. The weakly luminescent and water-soluble CuCP shows turn-on blue emission in the presence of histidine and histidine-rich peptides and proteins. The polymer can also stain histidine-rich proteins via gel electrophoresis. The limits of quantifications for histidine, glycine-histidine, serine-histidine, human serum albumin (HSA), bovine serum albumin, pepsin, trypsin, and lysozyme were found to be 300, 160, 600, 300, 600, 800, 120, and 290 nM, respectively. Utilizing the fluorescence turn-on property of CuCP, we measured HSA quantitatively in the urine samples. We also validated the present urinary HSA measurement assay with existing analytical techniques. Job's plot, 1H NMR, high-resolution mass spectrometry (HRMS), electron paramagnetic resonance (EPR), fluorescence, and UV-vis studies confirmed the ligand displacement from CuCP in the presence of histidine.

Theoretical structure determination of a class of heterocyclic compounds: The pyrazolidines

Theoretical calculations at the B3LYP-D3(BJ)/6–311++G(d,p) level of a series of 35 neutral pyrazolidines and 3 protonated derivatives have been carried out for different conformers generated using the CREST program. Seven optimized geometries were compared with X-ray determined structures and their 1H and 13C GIAO calculated chemical shifts with experimental results. The remaining pyrazolidines were compared with the available NMR information. The core of the present paper is the use of the pseudorotational wheel of Cremer and Pople to discuss the experimental and calculated conformation of the five-membered ring.

DSSP in GROMACS: Tool for Defining Secondary Structures of Proteins in Trajectories.

This work describes a fast implementation of a software algorithm associated with determination of protein secondary structure based on the Define Secondary Structure of Proteins (DSSP) algorithm. This implementation is fully compatible with the DSSP v.4 and DSSP v.2 algorithms and implemented as a native GROMACS trajectory analysis module, which allows us to analyze molecular dynamics trajectories without any restrictions of the original DSSP implementation. This implementation works much faster than the original DSSP v.4 and DSSP v.2 algorithms.