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Much of our current understanding of how small-molecule ligands interact with proteins stems from X-ray crystal structures determined at cryogenic (cryo) temperature. For proteins alone, room-temperature (RT) crystallography can reveal previously hidden, biologically relevant alternate conformations. However, less is understood about how RT crystallography may impact the conformational landscapes of protein-ligand complexes. Previously, we showed that small-molecule fragments cluster in putative allosteric sites using a cryo crystallographic screen of the therapeutic target PTP1B (Keedy et al., 2018). Here, we have performed two RT crystallographic screens of PTP1B using many of the same fragments, representing the largest RT crystallographic screens of a diverse library of ligands to date, and enabling a direct interrogation of the effect of data collection temperature on protein-ligand interactions. We show that at RT, fewer ligands bind, and often more weakly - but with a variety of temperature-dependent differences, including unique binding poses, changes in solvation, new binding sites, and distinct protein allosteric conformational responses. Overall, this work suggests that the vast body of existing cryo-temperature protein-ligand structures may provide an incomplete picture, and highlights the potential of RT crystallography to help complete this picture by revealing distinct conformational modes of protein-ligand systems. Our results may inspire future use of RT crystallography to interrogate the roles of protein-ligand conformational ensembles in biological function., Competing Interests: TS, JB, SA, EN, SH, LD, NP, AD, RT, DA, HO, Fv, DK No competing interests declared, (© 2023, Skaist Mehlman et al.)

Cerebral cavernous malformations (CCM) are dysplasias that primarily occur in the neurovasculature, and are associated with mutations in three genes: KRIT1, CCM2, and PDCD10, the protein products of which are KRIT1 (Krev/Rap1 Interaction Trapped 1; CCM1, cerebral cavernous malformations 1), CCM2 (cerebral cavernous malformations 2; OSM, osmosensing scaffold for MEKK3), and CCM3 (cerebral cavernous malformations 3; PDCD10, programmed cell death 10). Until recently, these proteins were relatively understudied at the molecular level, and only three folded domains were documented. These were a band 4.1, ezrin, radixin, moesin (FERM), and an ankyrin repeat domain (ARD) in KRIT1, and a phosphotyrosine-binding (PTB) domain in CCM2. Over the past 10 years, a crystallographic approach has been used to discover a series of previously unidentified domains within the CCM proteins. These include a non-functional Nudix (or pseudonudix) domain in KRIT1, a harmonin homology domain (HHD) in CCM2, and dimerization and focal adhesion targeting (FAT)-homology domains within CCM3. Many of the roles of these domains have been revealed by structure-guided studies that show the CCM proteins can directly interact with one another to form a signaling scaffold, and that the "CCM complex" functions in signal transduction by interacting with other binding partners, including ICAP1, RAP1, and MEKK3. In this chapter, we describe the crystallization of CCM protein domains alone, and with their interaction partners.

Transcription, catalyzed by RNA polymerase II (Pol II) in eukaryotes, is the first step in gene expression. RNA Pol II is a 12-subunit enzyme complex regulated by many different transcription factors during transcription initiation, elongation, and termination. During elongation, Pol II encounters various types of obstacles that can cause transcriptional pausing and arrest. Through decades of research on transcriptional pausing, it is widely known that Pol II can distinguish between different types of obstacles by its active site. A major class of obstacles is DNA lesions. While some DNA lesions can cause transient transcriptional pausing, which can be bypassed by Pol II itself or with the help from other elongation factors, bulky DNA damage can cause prolonged transcriptional pausing and arrest, which signals for transcription coupled repair. Using biochemical and structural biology approaches, the outcomes of many different types of DNA lesions, DNA modifications, and DNA binding molecules to transcription were studied. In this mini review, we will describe the in vitro transcription assays with Pol II to investigate the impacts of various DNA lesions on transcriptional outcomes and the crystallization method of lesion-arrested Pol II complex. These methods can provide a general platform for the structural and biochemical analysis of Pol II transcriptional pausing and bypass mechanisms., (Published by Elsevier Inc.)

Serial femtosecond crystallography (SFX) with an X-ray free-electron laser is used for the structural determination of proteins from a large number of microcrystals at room temperature. To examine the feasibility of pharmaceutical applications of SFX, a ligand-soaking experiment using thermolysin microcrystals has been performed using SFX. The results were compared with those from a conventional experiment with synchrotron radiation (SR) at 100 K. A protein-ligand complex structure was successfully obtained from an SFX experiment using microcrystals soaked with a small-molecule ligand; both oil-based and water-based crystal carriers gave essentially the same results. In a comparison of the SFX and SR structures, clear differences were observed in the unit-cell parameters, in the alternate conformation of side chains, in the degree of water coordination and in the ligand-binding mode.

The xyloglucan endotransglycosylase/hydrolase (XTH) gene family encodes enzymes of central importance to plant cell wall remodeling. The evolutionary history of plant XTH gene products is incompletely understood vis-à-vis the larger body of bacterial endoglycanases in Glycoside Hydrolase Family 16 (GH16). To provide molecular insight into this issue, high-resolution X-ray crystal structures and detailed enzyme kinetics of an extant transitional plant endoglucanase (EG) were determined. Functionally intermediate between plant XTH gene products and bacterial licheninases of GH16, Vitis vinifera EG16 (VvEG16) effectively catalyzes the hydrolysis of the backbones of two dominant plant cell wall matrix glycans, xyloglucan (XyG) and β(1,3)/β(1,4)-mixed-linkage glucan (MLG). Crystallographic complexes with extended oligosaccharide substrates reveal the structural basis for the accommodation of both unbranched, mixed-linked (MLG) and highly decorated, linear (XyG) polysaccharide chains in a broad, extended active-site cleft. Structural comparison with representative bacterial licheninases, a xyloglucan endotranglycosylase (XET), and a xyloglucan endohydrolase (XEH) outline the functional ramifications of key sequence deletions and insertions across the phylogenetic landscape of GH16. Although the biological role(s) of EG16 orthologs remains to be fully resolved, the present biochemical and tertiary structural characterization provides key insight into plant cell wall enzyme evolution, which will continue to inform genomic analyses and functional studies across species., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

Oligomerization by 3D domain swapping is found in a variety of proteins of diverse size, fold and function. In the early 1960s this phenomenon was postulated for the oligomers of ribonuclease A, but it was not until the 1990s that X-ray diffraction provided the first experimental evidence of this special manner of oligomerization. Nowadays, structural information has allowed the identification of these swapped oligomers in over one hundred proteins. Although the functional relevance of this phenomenon is not clear, this alternative folding of protomers into intertwined oligomers has been related to amyloid formation. Studies on proteins that develop 3D domain swapping might provide some clues on the early stages of amyloid formation. The SH3 domain is a small modular domain that has been used as a model to study the basis of protein folding. Among SH3 domains, the c-Src-SH3 domain emerges as a helpful model to study 3D domain swapping and amyloid formation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A detailed set of synthetic and crystallographic guidelines for the crystalline sponge method based upon the analysis of expediently synthesized crystal sponges using third-generation synchrotron radiation are reported. The procedure for the synthesis of the zinc-based metal-organic framework used in initial crystal sponge reports has been modified to yield competent crystals in 3 days instead of 2 weeks. These crystal sponges were tested on some small molecules, with two being unexpectedly difficult cases for analysis with in-house diffractometers in regard to data quality and proper space-group determination. These issues were easily resolved by the use of synchrotron radiation using data-collection times of less than an hour. One of these guests induced a single-crystal-to-single-crystal transformation to create a larger unit cell with over 500 non-H atoms in the asymmetric unit. This led to a non-trivial refinement scenario that afforded the best Flack x absolute stereochemical determination parameter to date for these systems. The structures did not require the use of PLATON/SQUEEZE or other solvent-masking programs, and are the highest-quality crystalline sponge systems reported to date where the results are strongly supported by the data. A set of guidelines for the entire crystallographic process were developed through these studies. In particular, the refinement guidelines include strategies to refine the host framework, locate guests and determine occupancies, discussion of the proper use of geometric and anisotropic displacement parameter restraints and constraints, and whether to perform solvent squeezing/masking. The single-crystal-to-single-crystal transformation process for the crystal sponges is also discussed. The presented general guidelines will be invaluable for researchers interested in using the crystalline sponge method at in-house diffraction or synchrotron facilities, will facilitate the collection and analysis of reliable high-quality data, and will allow construction of chemically and physically sensible models for guest structural determination.

Fenoxaprop‐P‐ethyl (FE) is widely applied as a post‐emergence aryloxyphenoxy propionate (AOPP) herbicide in agriculture. A novel FE hydrolase esterase from Acinetobacter sp. DL‐2 (AfeH) was identified which belongs to the family IV carboxylesterases and shows less than 30% identity to other reported homologues with known structure. In order to understand the catalytic mechanism, recombinant AfeH was prepared in Escherichia coli and crystallized using the sitting‐drop vapor‐diffusion method. X‐ray diffraction data were collected to 1.9 Å resolution. The crystal belonged to space group P1211, with unit‐cell parameters a = 84.27, b = 46.74, c = 258.68 Å. The Matthews coefficient (VM) was calculated to be 2.43 Å3 Da−1, which corresponds to a solvent content of 49.4%, suggesting the presence of three monomers in the crystallographic asymmetric unit. The crystal was assessed to be suitable for further structural determination, which is currently in progress. [ABSTRACT FROM AUTHOR]

Human 5′‐3′ exonuclease PLD3, a member of the phospholipase D family of enzymes, has been validated as a therapeutic target for treating Alzheimer's disease. Here, we have determined the crystal structure of the luminal domain of the enzyme at 2.3 Å resolution, revealing a bilobal structure with a catalytic site located between the lobes. We then compared the structure with published crystal structures of other human PLD family members which revealed that a number of catalytic and lipid recognition residues, previously shown to be key for phospholipase activity, are not conserved or, are absent. This led us to test whether the enzyme is actually a phospholipase. We could not measure any phospholipase activity but the enzyme shows robust nuclease activity. Finally, we have mapped key single nucleotide polymorphisms onto the structure which reveals plausible reasons as to why they have an impact on Alzheimer's disease. [ABSTRACT FROM AUTHOR]

The synthesis and characterization of the lithium m‐terphenylhydridoborates [Li(2,6‐Mes2C6H3)BH3]2 (1), Li(2,6‐Mes2C6H3)2BH2 (2) and Li(OEt2)(2,6‐Mes2C6H3)2BH2 (3) are reported. Hirshfeld Atom Refinement (HAR) of the experimentally obtained molecular structures by single‐crystal X‐ray crystallography allowed the determination of the exact positions of the hydrogen atoms. The bond situations of the various B−H⋅⋅⋅Li linkages were investigated by a complementary bonding analysis using various methods including atoms in molecules (AIM), electron localizability indicator (ELI−D), non‐covalent interaction (NCI) index and the compliance matrix. [ABSTRACT FROM AUTHOR]

A series of homoleptic rare earth (RE) complexes bearing phosphino-aryloxide ligands (1-RE, 2-La) has been prepared. The complexes have been characterised using multinuclear NMR and IR spectroscopy, X-ray crystallography and elemental analysis. Structural characterisation highlighted the different RE–P interactions as a result of differing Lewis acidity and ionic size across the series, hinting at the possibility of FLP-type activity. The potential reactivity of these complexes has been tested by reacting them with small molecules (H2, CO, CO2). A series of side-products (3-RE) has also been observed, isolated and characterised, featuring the incorporation of a phosphonium-aryloxide ligand. [ABSTRACT FROM AUTHOR]

We report the use of streaming data interfaces to perform fully online data processing for serial crystallography experiments, without storing intermediate data on disk. The system produces Bragg reflection intensity measurements suitable for scaling and merging, with a latency of less than 1 s per frame. Our system uses the CrystFEL software in combination with the ASAP::O data framework. In a series of user experiments at PETRA III, frames from a 16 megapixel Dectris EIGER2 X detector were searched for peaks, indexed and integrated at the maximum full-frame readout speed of 133 frames per second. The computational resources required depend on various factors, most significantly the fraction of non-blank frames (`hits'). The average single-thread processing time per frame was 242 ms for blank frames and 455 ms for hits, meaning that a single 96-core computing node was sufficient to keep up with the data, with ample headroom for unexpected throughput reductions. Further significant improvements are expected, for example by binning pixel intensities together to reduce the pixel count. We discuss the implications of real-time data processing on the `data deluge' problem from recent and future photon-science experiments, in particular on calibration requirements, computing access patterns and the need for the preservation of raw data.

The triethylammonium nitrate cocrystal of 5,5′-(triaz-1-ene-1,3-diyl)bis(3-nitro-1H-1,2,4-triazole), C4H3N11O4·C6H16N·NO3 (3), was obtained unintentionally as the product of an attempted synthesis of a heterocyclic C-bromonitrilimine from 3-amino-5-nitro-1,2,4-triazole (ANTA). In the solid state at 150 K, the structure of 3 is modulated by a pseudo-translation along [010] with Z* = 4 (Z = 8 in P1). Exact translational symmetry is broken by a slight modulation of the triazene molecules and nitrate anions, and by disorder of two of the four triethylammonium cations. An extensive hydrogen-bonding network connects the components of the structure and enables a relatively high density of 1.516 g cm−3. This feature, as well as its high nitrogen content, make this rare triazene-bridged 1,2,4-triazole and possible related similar compounds of interest as potential energetic materials.

Two enantiomeric non-heme iron complexes [Fe(R,R-BPMCN)(CH3CN)2](SbF6)2 and [Fe(S,S-BPMCN)(CH3CN)2](SbF6)2 (BPMCM = N,N′-dimethyl-N,N′-bis(pyridin-2-ylmethyl)-cyclohexane-1,2-diamine, C22H28N4) were obtained in parallel syntheses starting from the enantiomerically pure R,R and S,S BPMCN ligands. The FeII cations have a distorted octahedral FeN6 geometry formed by a chelating N,N,N,N-tetradentate BPMCN ligand and two molecules of acetonitrile. The ligand adopts a cis-α topology with the two pyridine groups coordinated trans to each other. In the crystals, a system of C—H...F hydrogen bonds links the cations to the hexafluoroantimonate anions, resulting in a three-dimensional architecture.

In their folded state, biomolecules exchange between multiple conformational states that are crucial for their function. Traditional structural biology methods, such as X-ray crystallography and cryogenic electron microscopy (cryo-EM), produce density maps that are ensemble averages, reflecting molecules in various conformations. Yet, most models derived from these maps explicitly represent only a single conformation, overlooking the complexity of biomolecular structures. To accurately reflect the diversity of biomolecular forms, there is a pressing need to shift toward modeling structural ensembles that mirror the experimental data. However, the challenge of distinguishing signal from noise complicates manual efforts to create these models. In response, we introduce the latest enhancements to qFit, an automated computational strategy designed to incorporate protein conformational heterogeneity into models built into density maps. These algorithmic improvements in qFit are substantiated by superior Rfree and geometry metrics across a wide range of proteins. Importantly, unlike more complex multicopy ensemble models, the multiconformer models produced by qFit can be manually modified in most major model building software (e.g., Coot) and fit can be further improved by refinement using standard pipelines (e.g., Phenix, Refmac, Buster). By reducing the barrier of creating multiconformer models, qFit can foster the development of new hypotheses about the relationship between macromolecular conformational dynamics and function.

MltG, a membrane-bound lytic transglycosylase, has roles in terminating glycan polymerization in peptidoglycan and incorporating glycan chains into the cell wall, making it significant in bacterial cell-wall biosynthesis and remodeling. This study provides the first reported MltG structure from Mycobacterium abscessus (maMltG), a superbug that has high antibiotic resistance. Our structural and biochemical analyses revealed that MltG has a flexible peptidoglycan-binding domain and exists as a monomer in solution. Further, the putative active site of maMltG was disclosed using structural analysis and sequence comparison. Overall, this study contributes to our understanding of the transglycosylation reaction of the MltG family, aiding the design of next-generation antibiotics targeting M. abscessus.

The absence of solvent molecules in high-resolution protein crystal structure models deposited in the Protein Data Bank (PDB) contradicts the fact that, for proteins crystallized from aqueous media, water molecules are always expected to bind to the protein surface, as well as to some sites in the protein interior. An analysis of the contents of the PDB indicated that the expected ratio of the number of water molecules to the number of amino-acid residues exceeds 1.5 in atomic resolution structures, decreasing to 0.25 at around 2.5 Å resolution. Nevertheless, almost 800 protein crystal structures determined at a resolution of 2.5 Å or higher are found in the current release of the PDB without any water molecules, whereas some other depositions have unusually low or high occupancies of modeled solvent. Detailed analysis of these depositions revealed that the lack of solvent molecules might be an indication of problems with either the diffraction data, the refinement protocol, the deposition process or a combination of these factors. It is postulated that problems with solvent structure should be flagged by the PDB and addressed by the depositors.

X-ray and neutron crystallography, as well as cryogenic electron microscopy (cryo-EM), are the most common methods to obtain atomic structures of biological macromolecules. A feature they all have in common is that, at typical resolutions, the experimental data need to be supplemented by empirical restraints, ensuring that the final structure is chemically reasonable. The restraints are accurate for amino acids and nucleic acids, but often less accurate for substrates, inhibitors, small-molecule ligands and metal sites, for which experimental data are scarce or empirical potentials are harder to formulate. This can be solved using quantum mechanical calculations for a small but interesting part of the structure. Such an approach, called quantum refinement, has been shown to improve structures locally, allow the determination of the protonation and oxidation states of ligands and metals, and discriminate between different interpretations of the structure. Here, we present a new implementation of quantum refinement interfacing the widely used structure-refinement software Phenix and the freely available quantum mechanical software ORCA. Through application to manganese superoxide dismutase and V- and Fe-nitrogenase, we show that the approach works effectively for X-ray and neutron crystal structures, that old results can be reproduced and structural discrimination can be performed. We discuss how the weight factor between the experimental data and the empirical restraints should be selected and how quantum mechanical quality measures such as strain energies should be calculated. We also present an application of quantum refinement to cryo-EM data for particulate methane monooxygenase and show that this may be the method of choice for metal sites in such structures because no accurate empirical restraints are currently available for metals.

Identifying and characterizing metal-binding sites (MBS) within macromolecular structures is imperative for elucidating their biological functions. CheckMyMetal (CMM) is a web based tool that facilitates the interactive validation of MBS in structures determined through X-ray crystallography and cryo-electron microscopy (cryo-EM). Recent updates to CMM have significantly enhanced its capability to efficiently handle large datasets generated from cryo-EM structural analyses. In this study, we address various challenges inherent in validating MBS within both X-ray and cryo-EM structures. Specifically, we examine the difficulties associated with accurately identifying metals and modeling their coordination environments by considering the ongoing reproducibility challenges in structural biology and the critical importance of well annotated, high-quality experimental data. CMM employs a sophisticated framework of rules rooted in the valence bond theory for MBS validation. We explore how CMM validation parameters correlate with the resolution of experimentally derived structures of macromolecules and their complexes. Additionally, we showcase the practical utility of CMM by analyzing a representative cryo-EM structure. Through a comprehensive examination of experimental data, we demonstrate the capability of CMM to advance MBS characterization and identify potential instances of metal misassignment.

Five chiral, racemic dialkyl α-hydroxyethylphosphonates and α-hydroxyethyl-diarylphosphine oxides were prepared in a scalable manner. Possibilities for the optical resolution of the racemic hydroxyphosphine oxides were explored via diastereomeric complex-forming experiments. The acidic calcium salt of O,O′-dibenzoyl-(2R,3R)-tartaric acid and O,O′-di-p-tolyl-(2R,3R)-tartaric acid were chosen as the resolving agents. The role of the solvent in the enantiomeric discrimination was investigated. The diastereomeric complex could be obtained in a crystalline form for α-hydroxyethyl-diphenylphosphine oxide and α-hydroxyethyl-bis(4-methylphenyl)phosphine oxide. However, in the third case, for α-hydroxyethyl-bis(3,5-dimethylphenyl)phosphine oxide, there was no chiral discrimination. Decomposition of the recrystallized diastereomeric complex followed by decomposition of the complex yielded the target compounds in 77/90% enantiomeric excess. The absolute configuration of the hydroxyethyl-diphenylphosphine oxide was determined by single-crystal X-ray diffraction measurements. The interactions stabilizing the supramolecular associate were evaluated. [ABSTRACT FROM AUTHOR]

ABSTRACT In this study, we report the synthesis, characterization, and biological investigation of antimony(III) halide complexes with various coordination architectures constructed from thiophene thiosemicarbazones. Antimony(III) thiophene‐2‐carbaldehyde thiosemicarbazone complexes (1, 2, 4, and 5) exhibit a square pyramidal geometry, with ligands coordinated to the central antimony atom in two distinct binding modes. In contrast, antimony(III) 2‐acetylthiophene thiosemicarbazone complexes (3, 6, and 7) adopt a seesaw geometry. These complexes (1–7) represent the first reported examples of antimony(III) halide thiosemicarbazone compounds. The unique coordination environments observed in these complexes are of significant importance within the realm of antimony chemistry. These synthesized complexes exhibit different coordination geometries as well as potential biological activities. The antiproliferative activity against the human breast adenocarcinoma (MCF‐7) cell line and antimicrobial activity against Gram‐positive and Gram‐negative bacteria were assessed. Antimony(III) thiophene‐2‐carbaldehyde thiosemicarbazone complexes (1, 2, 4, and 5) showed significant antiproliferative activity with IC50 values ranging from 8.5 to 19.1 μM, while antimony(III) 2‐acetylthiophene thiosemicarbazone complexes (3, 6, and 7) had higher IC50 values. Additionally, the antimony complexes demonstrated selective antimicrobial activity against Gram‐negative bacteria. [ABSTRACT FROM AUTHOR]

During the automatic processing of crystallographic diffraction experiments, beamstop shadows are often unaccounted for or only partially masked. As a result of this, outlier reflection intensities are integrated, which is a known issue. Traditional statistical diagnostics have only limited effectiveness in identifying these outliers, here termed Not‐Excluded‐unMasked‐Outliers (NEMOs). The diagnostic tool AUSPEX allows visual inspection of NEMOs, where they form a typical pattern: clusters at the low‐resolution end of the AUSPEX plots of intensities or amplitudes versus resolution. To automate NEMO detection, a new algorithm was developed by combining data statistics with a density‐based clustering method. This approach demonstrates a promising performance in detecting NEMOs in merged data sets without disrupting existing data‐reduction pipelines. Re‐refinement results indicate that excluding the identified NEMOs can effectively enhance the quality of subsequent structure‐determination steps. This method offers a prospective automated means to assess the efficacy of a beamstop mask, as well as highlighting the potential of modern pattern‐recognition techniques for automating outlier exclusion during data processing, facilitating future adaptation to evolving experimental strategies. [ABSTRACT FROM AUTHOR]

Pectocin M1 (PM1), the bacteriocin from phytopathogenic Pectobacterium carotovorum which causes soft rot disease, has a unique ferredoxin domain that allows it to use FusA of the plant ferredoxin uptake system. To probe the structure‐based mechanism of PM1 uptake, we determined the X‐ray structure of full‐length PM1, containing an N‐terminal ferredoxin and C‐terminal catalytic domain connected by helical linker, at 2.04 Å resolution. Based on published FusA structure and NMR data for PM1 ferredoxin domain titrated with FusA, we modeled docking of the ferredoxin domain with FusA. Combining the docking models with the X‐ray structures of PM1 and FusA enables us to propose the mechanism by which PM1 undergoes dynamic domain rearrangement to translocate across the target cell outer membrane. [ABSTRACT FROM AUTHOR]

Protein tyrosine phosphatase non‐receptor type 2 (PTPN2) has recently been recognized as a promising target for cancer immunotherapy. Despite extensive structural and functional studies of other protein tyrosine phosphatases, there is limited structural understanding of PTPN2. Currently, there are only five published PTPN2 structures and none are truly unbound due to the presence of a mutation, an inhibitor or a loop (related to crystal packing) in the active site. In this report, a novel crystal packing is revealed that resulted in a true apo PTPN2 crystal structure with an unbound active site, allowing the active site to be observed in a native apo state for the first time. Key residues related to accommodation in the active site became identifiable upon comparison with previously published PTPN2 structures. Structures of PTPN2 in complex with an established PTPN1 active‐site inhibitor and an allosteric inhibitor were achieved through soaking experiments using these apo PTPN2 crystals. The increased structural understanding of apo PTPN2 and the ability to soak in inhibitors will aid the development of future PTPN2 inhibitors. [ABSTRACT FROM AUTHOR]

The immunoglobulin (Ig)‐like domain is found in a broad range of proteins with diverse functional roles. While an essential β‐sandwich fold is maintained, considerable structural variations exist and are critical for functional diversity. The Rib‐domain family, primarily found as tandem‐repeat modules in the surface proteins of Gram‐positive bacteria, represents another significant structural variant of the Ig‐like fold. However, limited structural and functional exploration of this family has been conducted, which significantly restricts the understanding of its evolution and significance within the Ig superclass. In this work, a high‐resolution crystal structure of a Rib domain derived from the probiotic bacterium Limosilactobacillus reuteri is presented. This protein, while sharing significant structural similarity with homologous domains from other bacteria, exhibits a significantly increased thermal resistance. The potential structural features contributing to this stability are discussed. Moreover, the presence of two copper‐binding sites, with one positioned on the interface, suggests potential functional roles that warrant further investigation. [ABSTRACT FROM AUTHOR]

Carbonic anhydrase (CA) was among the first proteins whose X-ray crystal structure was solved to atomic resolution. CA proteins have essentially the same fold and similar active centers that differ in only several amino acids. Primary sulfonamides are well defined, strong and specific binders of CA. However, minor variations in chemical structure can significantly alter their binding properties. Over 1000 sulfonamides have been designed, synthesized and evaluated to understand the correlations between the structure and thermodynamics of their binding to the human CA isozyme family. Compound binding was determined by several binding assays: fluorescence-based thermal shift assay, stopped-flow enzyme activity inhibition assay, isothermal titration calorimetry and competition assay for enzyme expressed on cancer cell surfaces. All assays have advantages and limitations but are necessary for deeper characterization of these protein–ligand interactions. Here, the concept and importance of intrinsic binding thermodynamics is emphasized and the role of structure–thermodynamics correlations for the novel inhibitors of CA IX is discussed – an isozyme that is overexpressed in solid hypoxic tumors, and thus these inhibitors may serve as anticancer drugs. The abundant structural and thermodynamic data are assembled into the Protein–Ligand Binding Database to understand general protein–ligand recognition principles that could be used in drug discovery.

The assembly of the β-amyloid peptide (Aβ) to form oligomers and fibrils is closely associated with the pathogenesis and progression of Alzheimer's disease. Aβ is a shape-shifting peptide capable of adopting many conformations and folds within the multitude of oligomers and fibrils the peptide forms. These properties have precluded detailed structural elucidation and biological characterization of homogeneous, well-defined Aβ oligomers. In this paper, we compare the structural, biophysical, and biological characteristics of two different covalently stabilized isomorphic trimers derived from the central and C-terminal regions Aβ. X-ray crystallography reveals the structures of the trimers and shows that each trimer forms a ball-shaped dodecamer. Solution-phase and cell-based studies demonstrate that the two trimers exhibit markedly different assembly and biological properties. One trimer forms small soluble oligomers that enter cells through endocytosis and activate capase-3/7-mediated apoptosis, while the other trimer forms large insoluble aggregates that accumulate on the outer plasma membrane and elicit cellular toxicity through an apoptosis-independent mechanism. The two trimers also exhibit different effects on the aggregation, toxicity, and cellular interaction of full-length Aβ, with one trimer showing a greater propensity to interact with Aβ than the other. The studies described in this paper indicate that the two trimers share structural, biophysical, and biological characteristics with oligomers of full-length Aβ. The varying structural, assembly, and biological characteristics of the two trimers provide a working model for how different Aβ trimers can assemble and lead to different biological effects, which may help shed light on the differences among Aβ oligomers.

AbstractX-ray diffraction (XRD) is an analytical technique that has found several applications focusing on the identification of crystal structure, space groups, plane, and orientation, in addition to qualitative and quantitative phase identification, and polymorphism behavior. An XRD diffractogram pattern/Bragg’s peak can also provide valuable information that can be used for various food applications. While this review details the fundamental principles of XRD, the types of XRD systems, instrumentation, and the components thereof, the focus is to serve as a structured resource on explored applications of XRD in food, majorly revolving around food quality and safety. While recent studies relevant to the field are highlighted, leads for futuristic prospects are presented. With its unique approach, the XRD analysis can prove to be a rapid, robust, and sensitive nondestructive approach to food quality evaluation. Recent reports indicate its scope for nonconventional applications such as the assessment of 3D printability of foods, ice crystal formation, and screening food adulterants. Studies also highlight its scope to complement or replace conventional food quality testing approaches that involve the usage of chemicals, extensive sample preparation procedures, derivatization steps and demand long testing times. [ABSTRACT FROM AUTHOR]

Growing high‐quality crystals remains a necessary part of crystallography and many other techniques. This article tabulates and describes several techniques and variations that will help individuals grow high‐quality crystals in preparation for crystallographic techniques and other endeavors, such as form screening. The discussion is organized to focus on low‐tech approaches available in any laboratory. [ABSTRACT FROM AUTHOR]

Preparation of biomacromolecules for structural biology studies is a complex and time‐consuming process. The goal is to produce a highly concentrated, highly pure product that is often shipped to large facilities with tools to prepare the samples for crystallization trials or for measurements at synchrotrons and cryoEM centers. The aim of this article is to provide guidance and to discuss general considerations for shipping biomacromolecular samples. Details are also provided about shipping samples for specific experiment types, including solution‐ and cryogenic‐based techniques. These guidelines are provided with the hope that the time and energy invested in sample preparation is not lost due to shipping logistics. [ABSTRACT FROM AUTHOR]

BL19U1, an energy‐tunable protein complex crystallography beamline at the Shanghai Synchrotron Radiation Facility, has emerged as one of the most productive MX beamlines since opening to the public in July 2015. As of October 2023, it has contributed to over 2000 protein structures deposited in the Protein Data Bank (PDB), resulting in the publication of more than 1000 scientific papers. In response to increasing interest in structure‐based drug design utilizing X‐ray crystallography for fragment library screening, enhancements have been implemented in both hardware and data collection systems on the beamline to optimize efficiency. Hardware upgrades include the transition from MD2 to MD2S for the diffractometer, alongside the installation of a humidity controller featuring a rapid nozzle exchanger. This allows users to opt for either low‐temperature or room‐temperature data collection modes. The control system has been upgraded from Blu‐Ice to MXCuBE3, which supports website‐mode data collection, providing enhanced compatibility and easy expansion with new features. An automated data processing pipeline has also been developed to offer users real‐time feedback on data quality. [ABSTRACT FROM AUTHOR]

Boronic acids form diester bonds with cis-hydroxyl groups in carbohydrates. The formation of these adducts could impair the physical and chemical properties of precursors, even their biological activity. Two carbohydrate derivatives from d-fructose and d-arabinose and phenylboronic acid were synthesized in a straightforward one-step procedure and chemically characterized via spectroscopy and X-ray diffraction crystallography. Additionally, an acute toxicity test was performed to determine their lethal dose 50 (LD50) values by using Lorke's method. Analytical chemistry assays confirmed the formation of adducts by the generation of diester bonds with the β-d-pyranose of carbohydrates, including signals corresponding to the formation of new bonds, such as the stretching of B–O bonds. NMR spectra yielded information about the stereoselectivity in the synthesis reaction: Just one signal was found in the range for the anomeric carbon in the 13C NMR spectra of both adducts. The acute toxicity tests showed that the LD50 value for both compounds was 1265 mg/kg, while the effective dose 50 (ED50) for sedation was 531 mg/kg. However, differences were found in the onset and lapse of sedation. For example, the arabinose derivative induced sedation for more than 48 h at 600 mg/kg, while the fructose derivative induced sedation for less than 6 h at the same dose without the death of the mice. Thus, we report for the first time two boron-containing carbohydrate derivatives inducing sedation after intraperitoneal administration. They are bioactive and highly safe agents. Further biological evaluation is desirable to explore their medical applications. [ABSTRACT FROM AUTHOR]

This chapter discusses the use of diffraction simulators to improve experimental outcomes in macromolecular crystallography, in particular for future experiments aimed at diffuse scattering. Consequential decisions for upcoming data collection include the selection of either a synchrotron or free electron laser X-ray source, rotation geometry or serial crystallography, and fiber-coupled area detector technology vs. pixel-array detectors. The hope is that simulators will provide insights to make these choices with greater confidence. Simulation software, especially those packages focused on physics-based calculation of the diffraction, can help to predict the location, size, shape, and profile of Bragg spots and diffuse patterns in terms of an underlying physical model, including assumptions about the crystal's mosaic structure, and therefore can point to potential issues with data analysis in the early planning stages. Also, once the data are collected, simulation may offer a pathway to improve the measurement of diffraction, especially with weak data, and might help to treat problematic cases such as overlapping patterns.

Immunodominant membrane protein (IMP) is a prevalent membrane protein in phytoplasma and has been confirmed to be an F-actin-binding protein. However, the intricate molecular mechanisms that govern the function of IMP require further elucidation. In this study, the X-ray crystallographic structure of IMP was determined and insights into its interaction with plant actin are provided. A comparative analysis with other proteins demonstrates that IMP shares structural homology with talin rod domain-containing protein 1 (TLNRD1), which also functions as an F-actin-binding protein. Subsequent molecular-docking studies of IMP and F-actin reveal that they possess complementary surfaces, suggesting a stable interaction. The low potential energy and high confidence score of the IMP–F-actin binding model indicate stable binding. Additionally, by employing immunoprecipitation and mass spectrometry, it was discovered that IMP serves as an interaction partner for the phytoplasmal effector causing phyllody 1 (PHYL1). It was then shown that both IMP and PHYL1 are highly expressed in the S2 stage of peanut witches' broom phytoplasma-infected Catharanthus roseus. The association between IMP and PHYL1 is substantiated through in vivo immunoprecipitation, an in vitro cross-linking assay and molecular-docking analysis. Collectively, these findings expand the current understanding of IMP interactions and enhance the comprehension of the interaction of IMP with plant F-actin. They also unveil a novel interaction pathway that may influence phytoplasma pathogenicity and host plant responses related to PHYL1. This discovery could pave the way for the development of new strategies to overcome phytoplasma-related plant diseases.

The discovery of lytic polysaccharide monooxygenases (LPMOs), a family of copper-dependent enzymes that play a major role in polysaccharide degradation, has revealed the importance of oxidoreductases in the biological utilization of biomass. In fungi, a range of redox proteins have been implicated as working in harness with LPMOs to bring about polysaccharide oxidation. In bacteria, less is known about the interplay between redox proteins and LPMOs, or how the interaction between the two contributes to polysaccharide degradation. We therefore set out to characterize two previously unstudied proteins from the shipworm symbiont Teredinibacter turnerae that were initially identified by the presence of carbohydrate binding domains appended to uncharacterized domains with probable redox functions. Here, X-ray crystal structures of several domains from these proteins are presented together with initial efforts to characterize their functions. The analysis suggests that the target proteins are unlikely to function as LPMO electron donors, raising new questions as to the potential redox functions that these large extracellular multi-haem-containing c-type cytochromes may perform in these bacteria.

Recently, there have been numerous reports on the use of different fluorescent DNA stains for specific minor groove binding. The exploration of biological markers increases the safety of their use as diagnostic criteria. Single crystal analysis of DNA–ligand binding interactions is of essential importance to obtain the requirements for their usage in the pharmaceutical and medical industries. Dyes that bind to DNA, such as Hoechst 33342 or 4′,6-diamidino-2-phenylindole (DAPI), can be used not only for analytical use, but for medical purposes. DAPI and Hoechst 33342 are fluorescent dyes that bind to the minor groove of DNA, fluorescing brightly in the blue region with an emission maximum at approximately 461 nm when excited by ultraviolet light (~350 nm). This work focuses on the binding interactions of Hoechst 33342 with the specific DNA sequence d(CGTGAATTCACG)2. The structure of the complex was determined using single-crystal X-ray diffraction at a resolution of 1.9 Å in the space group P212121. The coordinates and structure factors are deposited in the RCSB Protein Data Bank (PDB) under entry 9FT8. The structure is nearly isomorphous with that of previously reported crystal structures of the oligonucleotide d(CGTGAATTCACG)2 alone (PDB ID: 5JU4) and with that in complexes with DAPI (5T4W). The adjustments in crystal interactions between the native DNA molecule and the DNA–DAPI complex are described. Hoechst 33342 selectively binded to the tight minor groove close to the midpoint of the B-DNA segment, adjacent to the A–T base pairs. It interacted with DNA through hydrogen bonding and van der Waals forces. The structural comparison revealed that Hoechst 33342 inserts itself in the minor groove in a strongly specific manner, displacing the ordered spine waters.

The ability of an octanuclear cubic coordination cage to catalyse a nucleophilic aromatic substitution reaction on a cavity‐bound guest was studied with 2,4‐dinitrofluorobenzene (DNFB) as the guest/substrate. It was found that DNFB undergoes a catalysed reaction with hydroxide ions within the cavity of the cubic cage (in aqueous buffer solution, pH 8.6). The rate enhancement of kcat/kuncat was determined to be 22, with cavity binding of the guest being required for catalysis to occur. The product, 2,4‐dinitrophenolate (DNP), remained bound within the cavity due to electrostatic stabilisation and exerts two apparently contradictory effects: it initially auto‐catalyses the reaction when present at low concentrations, but at higher concentrations inhibits catalysis when a pair of DNP guests block the cavity. When encapsulated, the UV/Vis absorption spectrum of DNP is red‐shifted when compared to the spectrum of free DNP in aqueous solution. Further investigations using other aromatic guests determined that a similar red‐shift on cavity binding also occurred for 4‐nitrophenolate (4NP) at pH 8.6. The red‐shift was used to determine the stoichiometry of guest binding of DNP and 4NP within the cage cavity, which was confirmed by structural analysis with X‐ray crystallography; and was also used to perform catalytic kinetic studies in the solution‐state. [ABSTRACT FROM AUTHOR]

Knowledge of hydrogen locations and protonation states is critical for a fundamental understanding of biological macromolecular function/interactions, and neutron macromolecular crystallography (NMX) is uniquely suited among the experimental structural-determination methods to provide this information. However, despite its potential, NMX remains a relatively niche technique, due to substantial limitations. This review explores NMX's role amongst the evolving landscape of structural biology, comparing and contrasting it to the historical gold standard of X-ray macromolecular crystallography (X-ray MX) and the increasingly prevalent electron-based methods—i.e., electron microscopy (EM) and electron diffraction (ED). Forthcoming developments (e.g., the European Spallation Source in Lund, Sweden, coming online) are expected to substantially address current limitations and ensure NMX will remain relevant in the coming decades. [ABSTRACT FROM AUTHOR]

Enzymes populate ensembles of structures necessary for catalysis that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography at an x-ray free electron laser to observe catalysis in a designed mutant isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations, and formation of the thioimidate intermediate selects for catalytically competent substates. The influence of cysteine ionization on the ICH ensemble is validated by determining structures of the enzyme at multiple pH values. Large molecular dynamics simulations in crystallo and time-resolved electron density maps show that Asp17 ionizes during catalysis and causes conformational changes that propagate across the dimer, permitting water to enter the active site for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics. [ABSTRACT FROM AUTHOR]

The title compound, N1,N2-dimethylethanedihydrazide, C4H10N4O2, was obtained by the methylation of oxalyl dihydrazide protected with phthalimide. The molecule is essentially non-planar with a dihedral angle between the two planar hydrazide fragments of 86.5 (2)°. This geometry contributes to the formation of a multi-contact three-dimensional supramolecular network via C—H...O, N—H...O and N—H...N hydrogen bonds.

The title compound, [Cu(HL)2(H2O)2] or [Cu(C4H4N3O2)2(H2O)2], is a mononuclear octahedral CuII complex based on 5-methyl-1H-1,2,4-triazole-3-carboxylic acid (H2L). [Cu(HL)2(H2O)2] was synthesized by reaction of H2L with copper(II) nitrate hexahydrate (2:1 stoichiometric ratio) in water under ambient conditions to produce clear light-blue crystals. The central Cu atom exhibits an N2O4 coordination environment in an elongated octahedral geometry provided by two bidentate HL− anions in the equatorial plane and two water molecules in the axial positions. Hirshfeld surface analysis revealed that the most important contributions to the surface contacts are from H...O/O...H (33.1%), H...H (29.5%) and H...N/N...H (19.3%) interactions.

Carotenoid cleavage dioxygenases (CCDs) constitute a superfamily of enzymes that are found in all domains of life where they play key roles in the metabolism of carotenoids and apocarotenoids as well as certain phenylpropanoids such as resveratrol. Interest in these enzymes stems not only from their biological importance but also from their remarkable catalytic properties including their regioselectivity, their ability to accommodate diverse substrates, and the additional activities (e.g., isomerase) that some of these enzyme possess. X-ray crystallography is a key experimental approach that has allowed detailed investigation into the structural basis behind the interesting biochemical features of these enzymes. Here, we describe approaches used by our lab that have proven successful in generating single crystals of these enzymes in resting or ligand-bound states for high-resolution X-ray diffraction analysis.

Critical assessment of structure prediction (CASP) conducts community experiments to determine the state of the art in computing protein structure from amino acid sequence. The process relies on the experimental community providing information about not yet public or about to be solved structures, for use as targets. For some targets, the experimental structure is not solved in time for use in CASP. Calculated structure accuracy improved dramatically in this round, implying that models should now be much more useful for resolving many sorts of experimental difficulties. To test this, selected models for seven unsolved targets were provided to the experimental groups. These models were from the AlphaFold2 group, who overall submitted the most accurate predictions in CASP14. Four targets were solved with the aid of the models, and, additionally, the structure of an already solved target was improved. An a posteriori analysis showed that, in some cases, models from other groups would also be effective. This paper provides accounts of the successful application of models to structure determination, including molecular replacement for X-ray crystallography, backbone tracing and sequence positioning in a cryo-electron microscopy structure, and correction of local features. The results suggest that, in future, there will be greatly increased synergy between computational and experimental approaches to structure determination.

The biological and functional significance of selected Critical Assessment of Techniques for Protein Structure Prediction 14 (CASP14) targets are described by the authors of the structures. The authors highlight the most relevant features of the target proteins and discuss how well these features were reproduced in the respective submitted predictions. The overall ability to predict three-dimensional structures of proteins has improved remarkably in CASP14, and many difficult targets were modeled with impressive accuracy. For the first time in the history of CASP, the experimentalists not only highlighted that computational models can accurately reproduce the most critical structural features observed in their targets, but also envisaged that models could serve as a guidance for further studies of biologically-relevant properties of proteins.

Enolase, a ubiquitous enzyme, catalyzes the reversible conversion of 2-phosphoglycerate (2PG) to phosphoenolpyruvate (PEP) in the glycolytic pathway of organisms of all three domains of life. The underlying mechanism of the 2PG to PEP conversion has been studied in great detail in previous work, however that of the reverse reaction remains to be explored. Here we present structural snapshots of Mycobacterium tuberculosis (Mtb) enolase in apo, PEP-bound and two 2PG-bound forms as it catalyzes the conversion of PEP to 2PG. The two 2PG-bound complex structures differed in the conformation of the bound product (2PG) viz the widely reported canonical conformation and a novel binding pose, which we refer to here as the alternate conformation. Notably, we observed two major differences compared with the forward reaction: the presence of MgB is non-obligatory for the reaction and 2PG assumes an alternate conformation that is likely to facilitate its dissociation from the active site. Molecular dynamics studies and binding free energy calculations further substantiate that the alternate conformation of 2PG causes distortions in both metal ion coordination and hydrogen-bonding interactions, resulting in an increased flexibility of the active-site loops and aiding product release. Taken together, this study presents a probable mechanism involved in PEP to 2PG catalysis that is likely to be mediated by the conformational change of 2PG at the active site.

The crystal structures of two single-enantiomer compounds, i.e. diphenyl [(R)-(+)-α-ethylbenzylamido]phosphate, C21H22NO3P or (C6H5O)2P(O)[NH-(R)-(+)CH(C2H5)(C6H5)] (I), and N-[(R)-(+)-α-ethylbenzyl]-P,P-diphenylphosphinic amide, C21H22NOP or (C6H5)2P(O)[NH-R-(+)CH(C2H5)(C6H5)] (II), were studied. The different environments at the phosphorus atoms, (O)2P(O)(N) and (C)2P(O)(N), allow the P=O/P—N bond strengths to be compared, as well as the N—H...O=P hydrogen-bond strengths, and P=O/N—H vibrations. The following characteristics related to diastereotopic C6H5O/C6H5 groups in I/II were considered: geometry parameters, contributions to the crystal packing, solution 13C/1H NMR chemical shifts, conformations, and NMR coupling constants. The phosphorus-carbon coupling constants nJPC (n = 2 and 3) in I and mJPC (m = 1, 2, 3 and 4) in II were evaluated. For a comparative study, chiral analogous structures were retrieved from the Cambridge Structural Database (CSD) and their geometries and conformations are discussed.

Perylene diimide (PDI) and naphthalene diimides (NDIs) are compounds widely used in supramolecular structures due to their versatile and functional properties. They have high absorptions and photoluminescence capabilities, which make them ideal for electronic transition studies. Reflux method, a widely employed synthetic technique, was utilized to synthesize NDI and PDI derivatives. In this method, the respective amino acids and NTDA (naphthalene‐1,4,5,8‐tetracarboxylic dianhydride) were combined in acetic acid and the resulting mixture was subjected to reflux. This study centered on a diverse set of NDI and PDI ligands, comprising L‐ala‐NDI, B‐ala‐NDI, Gly‐NDI, Imi‐NDI, Pyr‐NDI, L‐ala‐PDI, B‐ala‐PDI, Gly‐PDI, Imi‐PDI, and Pyr‐PDI ligands. Crystal structures were obtained for three NDI ligands, while the characterization of all ligands involved several analytical techniques such as NMR, IR, UV, DFT, TD‐DFT calculations, and single‐crystal x‐ray crystallography specifically for the NDI ligands. The investigation focused on studying the electron acceptor/donor behavior of the NDI and PDI ligands, identifying their potential for charge transfer applications. Furthermore, the NLO (nonlinear optical) response of all 10 NDI and PDI ligands was assessed through an analysis involving HOMO‐LUMO, TDM, EDDM, NCI, Iso‐surface, MEP, natural population, and DOS analysis. This evaluation encompassed the examination of linear polarizability, as well as first and second hyperpolarizability in the context of NLO. The findings of the study revealed that Gly‐PDI, Imi‐PDI, L‐ala‐PDI, and B‐ala‐PDI ligands displayed a higher NLO response compared with the other ligands. These results highlight the potential of these ligands for nonlinear optical applications. The comprehensive characterization and assessment of the NDI and PDI ligands contribute to a deeper understanding of their electron properties, positioning them as promising candidates for charge transfer and nonlinear optical materials. [ABSTRACT FROM AUTHOR]

Knowledge about the 3-dimensional structure, orientation and interaction of chemical compounds is important in many areas of science and technology. X-ray crystallography is one of the experimental techniques capable of providing a large amount of structural information for a given compound, and it is widely used for characterisation of organic and metal-organic molecules. The method provides precise 3D coordinates of atoms inside crystals, however, it does not directly deliver information about certain chemical characteristics such as bond orders, delocalization, charges, lone electron pairs or lone electrons. These aspects of a molecular model have to be derived from crystallographic data using refined information about interatomic distances and atom types as well as employing general chemical knowledge. This publication describes a curated automatic pipeline for the derivation of chemical attributes of molecules from crystallographic models. The method is applied to build a catalogue of chemical entities in an open-access crystallographic database, the Crystallography Open Database (COD). The catalogue of such chemical entities is provided openly as a derived database. The content of this catalogue and the problems arising in the fully automated pipeline are discussed, along with the possibilities to introduce manual data curation into the process. [ABSTRACT FROM AUTHOR]

The mechanism by which molecular oxygen is activated by the organic cofactor pyridoxal phosphate (PLP) for oxidation reactions remains poorly understood. Recent work has identified arginine oxidases that catalyze desaturation or hydroxylation reactions. Here, we investigate a desaturase from the Pseudoalteromonas luteoviolacea indolmycin pathway. Our work, combining X-ray crystallographic, biochemical, spectroscopic, and computational studies, supports a shared mechanism with arginine hydroxylases, involving two rounds of single-electron transfer to oxygen and superoxide rebound at the 4' carbon of the PLP cofactor. The precise positioning of a water molecule in the active site is proposed to control the final reaction outcome. This proposed mechanism provides a unified framework to understand how oxygen can be activated by PLP-dependent enzymes for oxidation of arginine and elucidates a shared mechanistic pathway and intertwined evolutionary history for arginine desaturases and hydroxylases.