Crystallography Experiments Research Articles

Hedgehog signalling in bone and osteoarthritis: the role of Smoothened and cholesterol.

Hedgehog signalling is essential for development, crucial for normal anatomical arrangement and activated during tissue damage repair. Dysregulation of hedgehog signalling is associated with cancer, developmental disorders and other diseases including osteoarthritis (OA). The hedgehog gene was first discovered in Drosophila melanogaster, and the pathway is evolutionarily conserved in most animals. Although there are several hedgehog ligands with different protein expression patterns, they share a common plasma membrane receptor, Patched1 and hedgehog signalling pathway activation is transduced through the G-protein-coupled receptor-like protein Smoothened (SMO) and downstream effectors. Functional assays revealed that activation of SMO is dependent on sterol binding, and cholesterol was observed bound to SMO in crystallography experiments. In vertebrates, hedgehog signalling coordinates endochondral ossification and balances osteoblast and osteoclast activation to maintain homeostasis. A recently discovered mutation of SMO in humans (SMOR173C ) is predicted to alter cholesterol binding and is associated with a higher risk of hip OA. Functional studies in mice and human tissue analysis provide evidence that hedgehog signalling is pathologically activated in chondrocytes of osteoarthritic cartilage.

Rational Design, Synthesis, and Mechanism of (3S,4R)-3-Amino-4-(difluoromethyl)cyclopent-1-ene-1-carboxylic Acid: Employing a Second-Deprotonation Strategy for Selectivity of Human Ornithine Aminotransferase over GABA Aminotransferase.

Human ornithine aminotransferase (hOAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that contains a similar active site to that of γ-aminobutyric acid aminotransferase (GABA-AT). Recently, pharmacological inhibition of hOAT was recognized as a potential therapeutic approach for hepatocellular carcinoma. In this work, we first studied the inactivation mechanisms of hOAT by two well-known GABA-AT inactivators (CPP-115 and OV329). Inspired by the inactivation mechanistic difference between these two aminotransferases, a series of analogues were designed and synthesized, leading to the discovery of analogue 10b as a highly selective and potent hOAT inhibitor. Intact protein mass spectrometry, protein crystallography, and dialysis experiments indicated that 10b was converted to an irreversible tight-binding adduct (34) in the active site of hOAT, as was the unsaturated analogue (11). The comparison of kinetic studies between 10b and 11 suggested that the active intermediate (17b) was only generated in hOAT and not in GABA-AT. Molecular docking studies and pKa computational calculations highlighted the importance of chirality and the endocyclic double bond for inhibitory activity. The turnover mechanism of 10b was supported by mass spectrometric analysis of dissociable products and fluoride ion release experiments. Notably, the stopped-flow experiments were highly consistent with the proposed mechanism, suggesting a relatively slow hydrolysis rate for hOAT. The novel second-deprotonation mechanism of 10b contributes to its high potency and significantly enhanced selectivity for hOAT inhibition.

Unraveling the Mechanics of a Repeat-Protein Nanospring: From Folding of Individual Repeats to Fluctuations of the Superhelix.

Tandem-repeat proteins comprise small secondary structure motifs that stack to form one-dimensional arrays with distinctive mechanical properties that are proposed to direct their cellular functions. Here, we use single-molecule optical tweezers to study the folding of consensus-designed tetratricopeptide repeats (CTPRs), superhelical arrays of short helix-turn-helix motifs. We find that CTPRs display a spring-like mechanical response in which individual repeats undergo rapid equilibrium fluctuations between partially folded and unfolded conformations. We rationalize the force response using Ising models and dissect the folding pathway of CTPRs under mechanical load, revealing how the repeat arrays form from the center toward both termini simultaneously. Most strikingly, we also directly observe the protein's superhelical tertiary structure in the force signal. Using protein engineering, crystallography, and single-molecule experiments, we show that the superhelical geometry can be altered by carefully placed amino acid substitutions, and we examine how these sequence changes affect intrinsic repeat stability and inter-repeat coupling. Our findings provide the means to dissect and modulate repeat-protein stability and dynamics, which will be essential for researchers to understand the function of natural repeat proteins and to exploit artificial repeats proteins in nanotechnology and biomedical applications.

Potential of Time-Resolved Serial Femtosecond Crystallography Using High Repetition Rate XFEL Sources

This perspective review describes emerging techniques and future opportunities for time-resolved serial femtosecond crystallography (TR-SFX) experiments using high repetition rate XFEL sources. High repetition rate sources are becoming more available with the European XFEL in operation and the recently upgraded LCLS-II will be available in the near future. One efficient use of these facilities for TR-SFX relies on pump–probe experiments using a laser to trigger a reaction of light-responsive proteins or mix-and-inject experiments for light-unresponsive proteins. With the view to widen the application of TR-SFX, the promising field of photocaged compounds is under development, which allows the very fast laser triggering of reactions that is no longer limited to naturally light-responsive samples. In addition to reaction triggering, a key concern when performing an SFX experiment is efficient sample usage, which is a main focus of new high repetition rate-compatible sample delivery methods.

Protein Fluctuations in Response to Random External Forces

Elastic network models (ENMs) have been widely used in the last decades to investigate protein motions and dynamics. There the intrinsic fluctuations based on the isolated structures are obtained from the normal modes of these elastic networks, and they generally show good agreement with the B-factors extracted from X-ray crystallographic experiments, which are commonly considered to be indicators of protein flexibility. In this paper, we propose a new approach to analyze protein fluctuations and flexibility, which has a more appropriate physical basis. It is based on the application of random forces to the protein ENM to simulate the effects of collisions of solvent on a protein structure. For this purpose, we consider both the Cα-atom coarse-grained anisotropic network model (ANM) and an elastic network augmented with points included for the crystallized waters. We apply random forces to these protein networks everywhere, as well as only on the protein surface alone. Despite the randomness of the directions of the applied perturbations, the computed average displacements of the protein network show a remarkably good agreement with the experimental B-factors. In particular, for our set of 919 protein structures, we find that the highest correlation with the B-factors is obtained when applying forces to the external surface of the water-augmented ANM (an overall gain of 3% in the Pearson’s coefficient for the entire dataset, with improvements up to 30% for individual proteins), rather than when evaluating the fluctuations obtained from the normal modes of a standard Cα-atom coarse-grained ANM. It follows that protein fluctuations should be considered not just as the intrinsic fluctuations of the internal dynamics, but also equally well as responses to external solvent forces, or as a combination of both.

ID23-2: an automated and high-performance microfocus beamline for macromolecular crystallography at the ESRF.

ID23-2 is a fixed-energy (14.2 keV) microfocus beamline at the European Synchrotron Radiation Facility (ESRF) dedicated to macromolecular crystallography. The optics and sample environment have recently been redesigned and rebuilt to take full advantage of the upgrade of the ESRF to the fourth generation Extremely Brilliant Source (ESRF-EBS). The upgraded beamline now makes use of two sets of compound refractive lenses and multilayer mirrors to obtain a highly intense (>1013 photons s-1) focused microbeam (minimum size 1.5 µm × 3 µm full width at half-maximum). The sample environment now includes a FLEX-HCD sample changer/storage system, as well as a state-of-the-art MD3Up high-precision multi-axis diffractometer. Automatic data reduction and analysis are also provided for more advanced protocols such as synchrotron serial crystallographic experiments.

3D printed devices and infrastructure for liquid sample delivery at the European XFEL.

The Sample Environment and Characterization (SEC) group of the European X-ray Free-Electron Laser (EuXFEL) develops sample delivery systems for the various scientific instruments, including systems for the injection of liquid samples that enable serial femtosecond X-ray crystallography (SFX) and single-particle imaging (SPI) experiments, among others. For rapid prototyping of various device types and materials, sub-micrometre precision 3D printers are used to address the specific experimental conditions of SFX and SPI by providing a large number of devices with reliable performance. This work presents the current pool of 3D printed liquid sample delivery devices, based on the two-photon polymerization (2PP) technique. These devices encompass gas dynamic virtual nozzles (GDVNs), mixing-GDVNs, high-viscosity extruders (HVEs) and electrospray conical capillary tips (CCTs) with highly reproducible geometric features that are suitable for time-resolved SFX and SPI experiments at XFEL facilities. Liquid sample injection setups and infrastructure on the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument are described, this being the instrument which is designated for biological structure determination at the EuXFEL.

Slipknot or Crystallographic Error: A Computational Analysis of the Plasmodium falciparum DHFR Structural Folds.

The presence of protein structures with atypical folds in the Protein Data Bank (PDB) is rare and may result from naturally occurring knots or crystallographic errors. Proper characterisation of such folds is imperative to understanding the basis of naturally existing knots and correcting crystallographic errors. If left uncorrected, such errors can frustrate downstream experiments that depend on the structures containing them. An atypical fold has been identified in P. falciparum dihydrofolate reductase (PfDHFR) between residues 20–51 (loop 1) and residues 191–205 (loop 2). This enzyme is key to drug discovery efforts in the parasite, necessitating a thorough characterisation of these folds. Using multiple sequence alignments (MSA), a unique insert was identified in loop 1 that exacerbates the appearance of the atypical fold-giving it a slipknot-like topology. However, PfDHFR has not been deposited in the knotted proteins database, and processing its structure failed to identify any knots within its folds. The application of protein homology modelling and molecular dynamics simulations on the DHFR domain of P. falciparum and those of two other organisms (E. coli and M. tuberculosis) that were used as molecular replacement templates in solving the PfDHFR structure revealed plausible unentangled or open conformations of these loops. These results will serve as guides for crystallographic experiments to provide further insights into the atypical folds identified.

Cover Picture: A {Ni12}‐Wheel‐Based Metal–Organic Framework for Coordinative Binding of Sulphur Dioxide and Nitrogen Dioxide (Angew. Chem. Int. Ed. 6/2022)

SO2 and NO2 are important air pollutants, and understanding the mechanism of capture materials drives the development of new clean-up technologies. In situ synchrotron X-ray crystallographic and spectroscopic experiments were used by Sihai Yang, Wei Shi et al. in their Research Article (e202115585) to establish a detailed molecular mechanism consisting of reversible coordination of SO2 and NO2 at the six open NiII sites on the unprecedented {Ni12}-wheel of a robust metal–organic framework material (NKU-100) at crystallographic resolution.

Implementation of wedged-serial protein crystallography at PROXIMA-1.

An approach for serial crystallography experiments based on wedged-data collection is described. This is an alternative method for recording in situ X-ray diffraction data on crystalline samples efficiently loaded in an X-ray compatible microfluidic chip. Proper handling of the microfluidic chip places crystalline samples at geometrically known positions with respect to the focused X-ray interaction area for serial data collection of small wedges. The integration of this strategy takes advantage of the greatly modular sample environment available on the endstation, which allows access to both in situ and more classical cryo-crystallography with minimum time loss. The method represents another optional data collection approach that adds up to the already large set of methods made available to users. Coupled with the advances in processing serial crystallography data, the wedged-data collection strategy proves highly efficient in minimizing the amount of required sample crystals for recording a complete dataset. From the advances in microfluidic technology presented here, high-throughput room-temperature crystallography experiments may become routine and should be easily extended to industrial use.

Best practices for time-resolved serial synchrotron crystallography.

With recent developments in X-ray sources, instrumentation and data-analysis tools, time-resolved crystallographic experiments, which were originally the preserve of a few expert groups, are becoming simpler and can be carried out at more radiation sources, and are thus increasingly accessible to a growing user base. However, these experiments are just that: discrete experiments, not just `data collections'. As such, careful planning and consideration of potential pitfalls is required to enable a successful experiment. Here, some of the key factors that should be considered during the planning and execution of a time-resolved structural study are outlined, with a particular focus on synchrotron-based experiments.

Unusual In‐Situ Preorganization and Postoxidation Steps Observed in Diels‐Alder Reactions of Styrylcyclohexene Dienes

AbstractUnusual products were obtained from cycloadditions reactions of two series of styrylcyclohexene dienes with dienophiles maleic anhydride and N‐phenylmaleimide. Experimental studies revealed that various dienes 3 underwent an initial in‐situ dehydration/double bond rearrangement prior to cycloaddition with maleic anhydride to give products 5 in 78–88 % yields. Alternatively, the cycloadducts of the reactions of 4 with N‐phenylmaleimide were hydoxylated stereoselectively in the same pot to give products 7, along with the usual adducts 6 in 83–89 % yields. Further, the reactions of the diene 4 with the dienophile dimethyl acetylenedicarboxylate were also studied, leading to the formation of the respective adducts 10 in 72–81 % yields. The stereochemistry of the new products was assigned based on their spectroscopic data and, in some cases, with X‐ray crystallography experiments.

Natural products repurposing of the H5N1-based lead compounds for the most fit inhibitors against 3C-like protease of SARS-CoV-2

Context: COVID-19 pandemic has caused more than 2.7 million mortality worldwide. Although the COVID-19 vaccine has been developed, the amount is still limited, and very few countries have reached ‘herd immunity’ level. In this regard, imported and community infections is still happening in the world. In order to complement the vaccine rollout, the drug is still necessary. Up to now, all the COVID-19 drugs in the market are still in emergency use, and their clinical application is still under tight surveillance. Thus, a breakthrough in drug development is necessary. Based on an extensive protein crystallography experiment, it is known that the 3C-like protease of SARS-CoV-2 plays an important role in the pathogenicity of the virus. Several inhibitors have been developed for this protein, including remdesivir that served as the standard in this experiment. However, recent findings in the wet lab also showed possible significant bioactivities for the repurposed influenza, and human rhinovirus leads for SARS-CoV-2. Previous research has developed flavonoid-based leads as H5N1 virus inhibitors. Aims: To develop lead compounds to inhibit 3C-like protease of SARS-CoV-2 from the existing H5N1 leads. Methods: The ligands and protein were prepared with energy minimization and the “add protonation” procedure. Then, the QSAR analysis was conducted to determine whether the ligands fit as leads for the 3C-like protease SARS-CoV-2 inhibitor. Molecular docking simulation was deployed for the selected ligand toward the 3C-like protease enzyme. Moreover, the molecular dynamics simulation was devised to examine the protein flexibility of the protease ligands. Results: It was found that only 9 out of the 19 repurposed H5N1-leads elicited significant QSAR-based properties for general antivirus, influenza antivirus, and antihuman rhinovirus bioactivities. In this regard, the leads were screened further with molecular docking, in silico ADME-TOX prediction, and molecular dynamics methods. Based on the further screen, the ligands of M00009235 and M00006834 were selected as lead compounds for 3C-like protease SARS-CoV-2 inhibitors. Conclusions: The ligands of M00009235 and M00006834 were selected as the best leads for inhibiting 3C-like protease of SARS-CoV-2 based on the virtual screening methods.

Identification and Structure-Activity Relationships of Dietary Flavonoids as Human Macrophage Migration Inhibitory Factor (MIF) Inhibitors.

Dietary flavonoids are known to have anti-inflammatory and anticancer effects, but their influences on human macrophage migration inhibitory factor (MIF), a vital proinflammatory cytokine recognized as a therapeutic target for infectious diseases and cancers, have been rarely reported. Here, we identified 24 dietary flavonoids that could inhibit the tautomerase activity of MIF, five of which exerted IC50 values lower than the positive control ISO-1 in the micromolar range: morin (IC50 = 11.01 ± 0.45 μM) and amentoflavone (IC50 = 13.32 ± 0.64 μM) exhibited the most potent efficacy followed by apigenin (IC50 = 42.74 ± 4.20 μM), naringin (IC50 = 51.38 ± 2.12 μM), and fisetin (IC50 = 51.99 ± 0.63 μM). X-ray crystallography, molecular docking, and cellular experiments were utilized to illustrate the molecular binding details and structure-activity relationships. Scaffold modifications of flavonoids significantly influenced the potency. What stands out for morin is the unique 2'-OH substitution. In addition, amentoflavone situated at the MIF trimer pore may impact MIF-CD74 signaling. The results also showed that flavonoids could suppress cell chemotaxis and nitric oxide production in RAW264.7 cells. Our results elucidate the molecular mechanism of flavonoids acting on MIF and shed light on developing lead compounds against MIF-involved diseases.

An electron diffractometer: a novel device for electron crystallography experiments

An electron diffractometer: a novel device for electron crystallography experiments

The upgrade of ID29: a novel instrument for time-resolved serial crystallography experiments at the ESRF Extremely Brilliant Source

The upgrade of ID29: a novel instrument for time-resolved serial crystallography experiments at the ESRF Extremely Brilliant Source

A SAXS-based approach to rationally evaluate radical scavengers - toward eliminating radiation damage in solution and crystallographic studies.

X-ray-based techniques are a powerful tool in structural biology but the radiation-induced chemistry that results can be detrimental and may mask an accurate structural understanding. In the crystallographic case, cryocooling has been employed as a successful mitigation strategy but also has its limitations including the trapping of non-biological structural states. Crystallographic and solution studies performed at physiological temperatures can reveal otherwise hidden but relevant conformations, but are limited by their increased susceptibility to radiation damage. In this case, chemical additives that scavenge the species generated by radiation can mitigate damage but are not always successful and the mechanisms are often unclear. Using a protein designed to undergo a large-scale structural change from breakage of a disulfide bond, radiation damage can be monitored with small-angle X-ray scattering. Using this, we have quantitatively evaluated how three scavengers commonly used in crystallographic experiments - sodium nitrate, cysteine, and ascorbic acid - perform in solution at 10°C. Sodium nitrate was the most effective scavenger and completely inhibited fragmentation of the disulfide bond at a lower concentration (500 µM) compared with cysteine (∼5 mM) while ascorbic acid performed best at 5 mM but could only reduce fragmentation by ∼75% after a total accumulated dose of 792 Gy. The relative effectiveness of each scavenger matches their reported affinities for solvated electrons. Saturating concentrations of each scavenger shifted fragmentation from first order to a zeroth-order process, perhaps indicating the direct contribution of photoabsorption. The SAXS-based method can detect damage at X-ray doses far lower than those accessible crystallographically, thereby providing a detailed picture of scavenger processes. The solution results are also in close agreement with what is known about scavenger performance and mechanism in a crystallographic setting and suggest that a link can be made between the damage phenomenon in the two scenarios. Therefore, our engineered approach might provide a platform for more systematic and comprehensive screening of radioprotectants that can directly inform mitigation strategies for both solution and crystallographic experiments, while also clarifying fundamental radiation damage mechanisms.

A new 2D Zn(II) coordination polymer as luminescent probe for highly selective detection of nitrofurazone

Coordination polymers (CPs) are peculiar category of materials having multidimensional framework and could be used as selective and sensitive sensors for ions and molecules. In this regard, a new Zn(II)-based coordination polymer (CP) [Zn(H2L)(H2O)2]n (1) has been derived from 1,3-bis(3,5-dicarboxylphenoxy)benzene (H4L) ligand and spectroscopically characterized alongwith single crystal X-ray diffraction. The crystallographic experiments reveal the existence of 2D layer in 1. The CP retains its framework integrity in water and exhibits luminescent property. This newly designed CP has been used as luminescent sensor to detect antibiotics by the decline in its luminescence intensity. The sensing experiments indicated that the CP displays sensitive and selective sensing against nitrofurazone (NZF) with limit of detection (LOD) of 1.12 ppm and Ksv value of 2.41 × 103 M−1. The experimentally observed decline in the luminescence intensity of 1 in presence of antibiotics has been explained with the help of theoretical calculations.

An Overview of the Proofreading Functions in Bacteria and in Severe Acute Respiratory Syndrome-Coronaviruses

Aim: To understand the structure-function relationship of the proofreading (PR) functions in eubacteria and viruses with special reference to Severe Acute Respiratory Syndrome-Coronaviruses (SARS-CoVs) and propose a plausible mechanism of action for PR exonucleases of SARS-CoVs.
 Study Design: Bioinformatics, biochemical, site-directed mutagenesis (SDM), X-ray crystallographic data were used to study the structure-function relationships of the PR exonucleases from bacteria and CoVs.
 Methodology: The protein sequences of the PR exonucleases of various DNA polymerases, and RNA polymerases of SARS, SARS-related and human CoVs (HCoVs) were obtained from PUBMED and SWISS-PROT databases. The advanced version of Clustal Omega was used for protein sequence analysis. Along with the conserved motifs identified by the bioinformatics analysis, the data already available by biochemical, SDM experiments and X-ray crystallographic analysis on these enzymes were used to arrive at the possible active amino acids in the PR exonucleases of these crucial enzymes.
 Results: A complete analysis of the active sites of the PR exonucleases from various bacteria and CoVs were done. The multiple sequence alignment (MSA) analysis showed many conserved amino acids, small and large peptide regions among them. Based on the conserved motifs, the PR exonucleases are found to fit broadly into two superfamilies, viz. DEDD and polymerase-histidinol phosphatase (PHP) superfamilies. The bacterial DNA polymerases I and II, RNase D, RNase T and ε-subunit of DNA polymerases III belong to the DEDD superfamily. The PR enzymes from SARS, SARS-related CoVs and other HCoVs also essentially belong to the DEDD superfamily. The DEDD superfamily either uses an invariant Tyr or a His as proton acceptor during catalysis. Depending on the proton acceptor, they are further classified into DEDHD and DEDYD subfamilies. RNase T, ε-subunit of DNA polymerases III and the SARS, SARS-related CoVs and other HCoVs belong to DEDHD subfamily. However, the SARS, SARS-related CoVs and other HCoVs showed additional zinc finger motifs (ZFMs) in their active sites. DNA polymerases I, II and RNase D belong to DEDYD subfamily. The bacterial DNA polymerases X, YcdX phosphoesterases and the co-editing exonuclease of DNA polymerases III belong to the PHP superfamily. Based on the MSA, X-ray crystallographic analyses and SDM experiments, the proposed active-site proton acceptor is Tyr/His in DEDDY/H subfamilies and His in PHP superfamily of PR exonucleases. 
 Conclusions: Based on the similarities of active site amino acids/motifs, it may be concluded that the DEDD and PHP superfamilies of PR exonucleases should have evolved from a common ancestor but diverged very long ago. The biochemical properties of these enzymes, including the four conserved acidic amino acid residues in the catalytic core, suggest that the CoVs might have acquired the exonuclease function, possibly from a prokaryote. However, the presence of two zinc fingers in the PR active site of the SARS, SARS-related CoVs and other HCoVs sets their PR exonucleases apart from other homologues.

Low-background serial crystallography experiments

Low-background serial crystallography experiments