Determining Protein Structures Using X-Ray Crystallography.

X-ray crystallography is a robust and widely used technique that facilitates the three-dimensional structure determination of proteins at an atomic scale. This methodology entails the growth of protein crystals under controlled conditions followed by their exposure to X-ray beams and the subsequent analysis of the resulting diffraction patterns via computational tools to determine the three-dimensional architecture of the protein. However, achieving high-resolution structures through X-ray crystallography can be quite challenging due to complexities associated with protein purity, crystallization efficiency, and crystal quality.In this chapter, we provide a detailed overview of the gene to structure determination pipeline used in X-ray crystallography, a crucial tool for understanding protein structures. The chapter covers the steps in protein crystallization, along with the processes of data collection, processing, structure determination, and refinement. The most commonly faced challenges throughout this procedure are also addressed. Finally, the importance of standardized protocols for reproducibility and accuracy is emphasized, as they are crucial for advancing the understanding of protein structure and function.

Crystallization and In Situ Room Temperature Data Collection Using the Crystallization Facility at Harwell and Beamline VMXi, Diamond Light Source.

Protocols for robotic protein crystallization using the Crystallization Facility at Harwell and in situ room temperature data collection from crystallization plates at Diamond Light Source beamline VMXi are described. This approach enables high-quality room-temperature crystal structures to be determined from multiple crystals in a straightforward manner and provides very rapid feedback on the results of crystallization trials as well as enabling serial crystallography. The value of room temperature structures in understanding protein structure, ligand binding, and dynamics is becoming increasingly recognized in the structural biology community. This pipeline is accessible to users from all over the world with several available modes of access. Crystallization experiments that are set up can be imaged and viewed remotely with crystals identified automatically using a machine learning tool. Data are measured in a queue-based system with up to 60° rotation datasets from user-selected crystals in a plate. Data from all the crystals within a particular well or sample group are automatically merged using xia2.multiplex with the outputs straightforwardly accessed via a web browser interface.

Fragility and Tendency to Crystallization for Structurally Related Compounds.

The present study was designed to investigate the physical stability of three organic materials with similar chemical structures. The examined compounds revealed completely different crystallization tendencies in their supercooled liquid states and were classified into three distinct classes based on their tendency to crystallize. (S)-4-Benzyl-2-oxazolidinone easily crystallizes during cooling from the melt; (S)-4-Benzylthiazolidine-2-thione does not crystallize during cooling from the melt, but crystallizes easily during subsequent reheating above Tg; and (S)-4-Benzyloxazolidine-2-thione does not crystallize either during cooling from the melt or during reheating. Such different tendencies to crystallize are observed despite the very similar chemical structures of the compounds, which only differ in oxide or sulfur atoms in one of their rings. We also studied the isothermal crystallization kinetics of the materials that were shown to transform into a crystalline state. Molecular dynamics and thermal properties were thoroughly investigated using broadband dielectric spectroscopy, as well as conventional and temperature-modulated differential scanning calorimetry in the wide temperature range. It was found that all three glass formers have the same dynamic fragility (m = 93), calculated directly from dielectric structural relaxation times. This result verifies that dynamic fragility is not related to the tendency to crystallize. In addition, thermodynamic fragility predictions were also made using calorimetric data. It was found that the thermodynamic fragility evaluated based on the width of the glass transition, observed in the temperature dependence of heat capacity, correlates best with the tendency to crystallize.

Efficient in situ screening of and data collection from microcrystals in crystallization plates.

A considerable bottleneck in serial crystallography at XFEL and synchrotron sources is the efficient production of large quantities of homogenous, well diffracting microcrystals. Efficient high-throughput screening of batch-grown microcrystals and the determination of ground-state structures from different conditions is thus of considerable value in the early stages of a project. Here, a highly sample-efficient methodology to measure serial crystallography data from microcrystals by raster scanning within standard in situ 96-well crystallization plates is described. Structures were determined from very small quantities of microcrystal suspension and the results were compared with those from other sample-delivery methods. The analysis of a two-dimensional batch crystallization screen using this method is also described as a useful guide for further optimization and the selection of appropriate conditions for scaling up microcrystallization.

Influence of Nucleation on Relaxation, Molecular Cooperativity, and Physical Stability of Celecoxib Glass.

Although nucleation is considered the first step in the crystallization of glass materials, the structure and properties of the nuclei are not understood well. Influence of nucleation on the structure and dynamics of celecoxib glass was evaluated in this study. The nuclei for Form III were induced by annealing the glass at freezing temperature, and their impact on the relaxation behavior was investigated using thermal analysis and broadband dielectric spectroscopy to find accelerated α relaxation and suppressed ß relaxation. In addition, observed after nucleation was a decrease in cooperativity of the molecular motion, presumably because of the appearance of void spaces in the glass structure. During long-term isothermal crystallization studies, crystal growth to Form III was accelerated in the presence of the nuclei, whereas this effect was less remarkable when a different crystal form dominated the crystallization behavior. These observations should provide more detailed insights into the nucleation mechanism and impact of nucleation on molecular dynamics including physical stability of pharmaceutical glasses. In addition, discussed is the remarkable acceleration of the crystallization rate of the celecoxib glass just below its Tg, which could be understood by diffusionless crystal growth.

Preparation and formulation of progesterone para-aminobenzoic acid co-crystals with improved dissolution and stability.

The crystal structure of a new Progesterone (PROG) co-crystal with para-aminobenzoic acid (PABA) showing enhanced solution properties is reported. PROG-PABA co-crystal was first identified though an in silico coformer screening process using the CSD Co-crystal deign function, then confirmed through a solution evaporation crystallisation experiment. The resulting co-crystal was characterized using single crystal X-ray diffraction, differential scanning calorimetry and Fourier-transform infrared spectroscopy. Liquid assisted grinding was selected as a suitable scale up method compared to spray drying and antisolvent methods due to minimal starting material phases in the final product. Following scale up, aqueous solubility, stability and dissolution measurements were carried out. PROG-PABA showed increased distinct aqueous solubility and dissolution compared to PROG starting material and was shown to be stable at 75 % relative humidity for 3 months. Tablets containing co-crystal were produced then compared to the Utrogestan® soft gel capsule formulation through a dissolution experiment. PROG-PABA tablets showed a substantial increase in dissolution over the course of the experiment with over 30× the amount of PROG dissolved at the 3-hour time point. This co-crystal shows positive implications for developing an improved oral PROG formulation.

Devitrite (Na2Ca3Si6O16) phase dominated nanostructured 45S5 bioactive glass: exploring its structural and biological properties.

This research study is primarily centred around calcination temperature and time influence on phase formation in bioactive glasses (BGs). In the present study, BG with a nominal composition of 45S5 was synthesized through the sol-gel process. The developed BGs then underwent heat treatment for various sintering durations and temperatures. X-ray diffraction (XRD) patterns of the BGs reveals that the sintering process led to the crystallization of both devitrite (Na2Ca3Si6O16) and combeite (Na2Ca2Si3O9) phases. The field emission scanning electron microscopy study divulges morphological alterations, from sheet-like to rod-like structures to eventually transforming into spherical and sheet-like structures. The surface area and Type-IV mesoporous porosity were validated through Brunauer Emmett Teller analysis, highlighting a notable increase in pore volume and mechanical strength at a lower sintering temperature.In vitroapatite formation was carried out in Hank's balance salt in order to evaluate the bioactivity of the glass. After 7 d of immersion in simulated body fluid (SBF), XRD patterns and scanning electron microscopy micrographs results showed that formation of hydroxyapatite layer on the surface of the BGs. The BG compatibility with erythrocytes (red blood cells) was also studied, and the results revealed that there was only a low 2% lysis, showing good hemocompatibility. The drug loading and release behaviour of the BGs was studied in thein vitroanalysis. The findings showed a high drug encapsulation effectiveness of up to 90% and continuous drug release from the BGs for 24 h. The materials biocompatibility was unambiguously confirmed by cytocompatibility and proliferation studies. This study provides compelling evidence for the exceptional efficacy and promise of the distinct 45S5 BGs in advancing the field of regenerative medicine.

Phase Identification and Discovery of an Elusive Polymorph of Drug-Polymer Inclusion Complex Using Automated 3D Electron Diffraction.

3D electron diffraction (3D ED) has shown great potential in crystal structure determination in materials, small organic molecules, and macromolecules. In this work, an automated, low-dose and low-bias 3D ED protocol has been implemented to identify six phases from a multiple-phase melt-crystallisation product of an active pharmaceutical ingredient, griseofulvin (GSF). Batch data collection under low-dose conditions using a widely available commercial software was combined with automated data analysis to collect and process over 230 datasets in three days. Accurate unit cell parameters obtained from 3D ED data allowed direct phase identification of GSF Forms III, I and the known GSF inclusion complex (IC) with polyethylene glycol (PEG) (GSF-PEG IC-I), as well as three minor phases, namely GSF Forms II, V and an elusive new phase, GSF-PEG IC-II. Their structures were then directly determined by 3D ED. Furthermore, we reveal how the stabilities of the two GSF-PEG IC polymorphs are closely related to their crystal structures. These results demonstrate the power of automated 3D ED for accurate phase identification and direct structure determination of complex, beam-sensitive crystallisation products, which is significant for drug development where solid form screening is crucial for the overall efficacy of the drug product.

Water-mediated phase transformations of posaconazole: An intricate jungle of crystal forms.

Posaconazole is a broad-spectrum antifungal agent exhibiting rich polymorphism. Up to now, a total of fourteen different crystal forms have been reported, sometimes with an ambiguous nomenclature, but less is known about their properties and stability relationships. Investigating the solid-state of a drug compound is essential to identify the most stable form under working conditions and to prevent the risk of undesired solid-phase transformations under processing and storage. In this paper, we study posaconazole polymorphism by providing a description of its polymorphs, hydrates, and solvates. Powder X-ray diffraction (PXRD), dynamic vapor sorption (DVS), spectroscopic and thermal techniques were employed to characterize the different forms. In addition, the solid-phase transformations of posaconazole in aqueous suspensions were studied by means of Raman microscopy. Surprisingly, we found that Form S, the crystal form contained in the marketed oral suspension, is not the most stable form in water. Form S readily converts to a more stable hydrate, i.e. Form A, after storage in water for two weeks. In the commercial oral formulation the conversion between the two forms is prevented by the presence of polysorbate 80. Such insights into the stabilizing excipient effects beyond particle dispersion are critical to formulators.

Crystal size, a key character of lactose crystallization affecting microstructure, surface chemistry and reconstitution of milk powder.

Lactose crystallization during storage deteriorates reconstitution performance of milk powders, but the relationship between lactose crystallization and reconstitution is inexplicit. The objective of this study is to characterize crystalline lactose in the context of formulation and elucidate the complex relationship between lactose crystallization and powder functionality. Lactose in Skim Milk Powder (SMP), Whole Milk Powder (WMP) and Fat-Filled Milk Powder (FFMP) stored under 23 %, 53 % and 75 % Relative Humidity (RH) at 25  â„ƒ for four months was compared. Lactose, surface chemistry and microstructure of FFMP stored at 25 â„ƒ and 40 â„ƒ at 23 % to 75 % RH for four months were also analyzed and interpreted. At the same RH, FFMP crystallized in the same pattern as WMP. At 53 % RH, FFMP and WMP differentiated from SMP in terms of lactose morphology as well as the ratio between anhydrous α-lactose and anhydrous ß-lactose. Lactose remained amorphous at 23 % RH, crystallized predominantly to α/ß-lactose (1:4) at 40 to 58 % RH and to α-lactose monohydrate at 75 % RH. The crystallinity index was similar for all powders containing crystalline lactose. The estimated crystallite size increased from approx. 0.1 to 20 µm with increasing RH and temperature. When amorphous lactose crystallized into crystals below approx. 0.1 µm at 25 °C and 43 % RH, the microstructure and surface lipid were comparable to that of the reference powder. This powder reconstituted into a stable suspension system comparable to that of reference (well performing) powders. These results demonstrate that crystallite size is the key property linking lactose crystallization and reconstitution. Our finding thus indicates limiting crystallite size is important for maintaining desired product quality.

Preparation, Characterization, in-vitro and in-vivo Pharmacokinetic Evaluation of Thermostable Dimethyl Fumarate Cocrystals.

Dimethyl fumarate (DMF) is an FDA-approved drug for treating relapsing-remitting multiple sclerosis; but it is susceptible to sublimation leading to its loss during processing. Cocrystals can protect against thermal energy via the interaction of DMF with a coformer via weak forces of interaction. With this hypothesis, we have, for the first time, prepared DMF cocrystals using the solvent evaporation method using coformers like citric acid and succinic acid screened by in-silico predictions and hydrogen bonding properties. Analysis using infra-red (IR), powder x-ray diffraction (PXRD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and sublimation evaluation characterized cocrystals and their thermostability. Comparative analysis of the release profile has been done by dissolution and pharmacokinetic study of DMF and its cocrystals. The cocrystals have improved thermal stability and better pharmacological activities than DMF. In the safety and efficacy evaluation of the formulated cocrystals, they were found to be non-cytotoxic, antioxidant, and inhibiting IL-6 and TNF-α in PBMC induced by lipopolysaccharide (LPS). We have obtained cocrystals of DMF with improved thermal stability and better pharmacological activities than DMF.

Engineered antigen-binding fragments for enhanced crystallization of antibody:antigen complexes.

The atomic-resolution structural information that X-ray crystallography can provide on the binding interface between a Fab and its cognate antigen is highly valuable for understanding the mechanism of interaction. However, many Fab:antigen complexes are recalcitrant to crystallization, making the endeavor a considerable effort with no guarantee of success. Consequently, there have been significant steps taken to increase the likelihood of Fab:antigen complex crystallization by altering the Fab framework. In this investigation, we applied the surface entropy reduction strategy coupled with phage-display technology to identify a set of surface substitutions that improve the propensity of a human Fab framework to crystallize. In addition, we showed that combining these surface substitutions with previously reported Crystal Kappa and elbow substitutions results in an extraordinary improvement in Fab and Fab:antigen complex crystallizability, revealing a strong synergistic relationship between these sets of substitutions. Through comprehensive Fab and Fab:antigen complex crystallization screenings followed by structure determination and analysis, we defined the roles that each of these substitutions play in facilitating crystallization and how they complement each other in the process.

Pharmaceutical co-crystals: A green way to enhance drug stability and solubility for improved therapeutic efficacy.

Pharmaceutical co-crystals have gained significant attention in recent years as a promising green and sustainable method for poorly soluble drugs to improve their solubility, stability, and bioavailability. In the drug development research field, it is an extremely useful technique as it does not require a large number of synthetic steps as well a minimum amount of solvent is utilized or sometimes without solvent. This review presents a comprehensive investigation into the design, synthesis, characterization, and evaluation of pharmaceutical co-crystals. The study focuses on exploring different strategies for co-crystal formation, including co-grinding, solvent evaporation, and liquid-assisted grinding. Various characterization techniques such as SCXRD, PXRD, FTIR, and DSC were employed to confirm the formation and structural features of the co-crystals. The article also highlights the significance of understanding the intermolecular interactions within co-crystals and their influence on physicochemical properties. Furthermore, the article discusses the potential applications of pharmaceutical co-crystals in enhancing drug solubility, dissolution rate, and oral bioavailability, leading to improved therapeutic efficacy. Overall, this review provides valuable insights into the design and development of pharmaceutical co-crystals, offering a promising avenue for overcoming the difficulties brought on by poorly soluble drugs.

Competition between homogeneous and heterogeneous crystallization of CaCO3 during water softening.

Homogeneous and heterogeneous crystallization of CaCO3 simultaneously occur in seed-induced crystallization during water softening, while suppressing homogeneous crystallization is necessary due to the production of fine particulates that poorly precipitate. However, homogeneous crystallization is difficult to distinguish from heterogeneous crystallization. Consequently, a central focus in improving water softening is understanding their competing activities. In this study, a novel method for distinguishing homogeneous and heterogeneous calcium carbonate crystallization is described that utilizes magnetite as seed particles. Results showed that saturation index (SI) was the primary driver of both homogeneous and heterogeneous crystallizations. Heterogeneous crystallization was preferentially promoted at low SI, while homogeneous crystallization was promoted at high SI. The highest suppression effect to homogeneous crystallization occurred at SI of about 1.01. Seed dosage and mean particle size were the primary parameters related to the competition of the crystallization types. Higher seed dosage and smaller seed particle sizes promoted heterogeneous crystallization and suppressed homogeneous crystallization. Due to the good adaptability of heterogeneous crystallization at low SI, the absorption of CO2 from the air into the solutions also improved the efficiency of hardness removal. The introduction of seed particles did not change crystalline product phases, with calcite being the only observed phase and possessing rhombohedral forms with highly regular and smooth edges. Water softening pilot test results showed that SI of 1.5 was more favorite for CaCO3 layer formation on seed surface and hardness removal in comparison with SI of 1.0 and 2.0. Overall, the results from this study demonstrate that the introduction of seed particles is a promising approach to suppress the homogeneous crystallization of CaCO3. Moreover, these results can serve as a framework for improved seed-induced crystallization during water softening.

Quabodepistat (OPC-167832), a Novel Antituberculosis Drug Candidate: Enhancing Oral Bioavailability via Cocrystallization and Mechanistic Analysis of Bioavailability in Two Cocrystals.

Quabodepistat (code name OPC-167832) is a novel antituberculosis drug candidate. This study aimed to discover cocrystals that improve oral bioavailability and to elucidate the mechanistic differences underlying the bioavailability of different cocrystals. Screening yielded two cocrystals containing 2,5-dihydroxybenzoic acid (2,5DHBA) or 2-hydroxybenzoic acid (2HBA). In bioavailability studies in beagle dogs, both cocrystals exhibited better bioavailability than the free form; however, the extent of bioavailability of cocrystals with 2HBA (quabodepistat-2HBA) was 1.4-fold greater than that of cocrystals with 2,5DHBA (quabodepistat-2,5DHBA). Dissolution studies at pH 1.2 yielded similar profiles for both cocrystals, although the percent dissolution differed: quabodepistat-2HBA dissolved more slowly than quabodepistat-2,5DHBA. The poor solubility of quabodepistat-2HBA is likely the primary factor limiting dissolution at pH 1.2. To identify a dissolution method that maintains the bioavailability in beagle dogs, we performed pH-shift dissolution studies that mimic the dynamic pH change from the stomach to the small intestine. Quabodepistat-2HBA demonstrated supersaturation after the pH was increased to 6.8, while quabodepistat-2,5DHBA did not demonstrate supersaturation. This result was consistent with the results of bioavailability studies in beagle dogs. We conclude that a larger quantity of orally administered quabodepistat-2HBA remained in its cocrystal form while being transferred to the small intestine compared with quabodepistat-2,5DHBA.

High-throughput nanoscale crystallization of dihydropyridine active pharmaceutical ingredients.

Single-crystal X-ray diffraction analysis of small molecule active pharmaceutical ingredients is a key technique in the confirmation of molecular connectivity, including absolute stereochemistry, as well as the solid-state form. However, accessing single crystals suitable for X-ray diffraction analysis of an active pharmaceutical ingredient can be experimentally laborious, especially considering the potential for multiple solid-state forms (solvates, hydrates and polymorphs). In recent years, methods for the exploration of experimental crystallization space of small molecules have undergone a `step-change', resulting in new high-throughput techniques becoming available. Here, the application of high-throughput encapsulated nanodroplet crystallization to a series of six dihydropyridines, calcium channel blockers used in the treatment of hypertension related diseases, is described. This approach allowed 288 individual crystallization experiments to be performed in parallel on each molecule, resulting in rapid access to crystals and subsequent crystal structures for all six dihydropyridines, as well as revealing a new solvate polymorph of nifedipine (1,4-dioxane solvate) and the first known solvate of nimodipine (DMSO solvate). This work further demonstrates the power of modern high-throughput crystallization methods in the exploration of the solid-state landscape of active pharmaceutical ingredients to facilitate crystal form discovery and structural analysis by single-crystal X-ray diffraction.

Amorphization of different furosemide polymorphic forms during ball milling: Tracking solid-to-solid phase transformations.

Ball milling is used, not only to reduce the particle size of pharmaceutical powders, but also to induce changes in the physical properties of drugs. In this work we prepared three crystal forms of furosemide (forms Ⅰ, Ⅱ, and Ⅲ) and studied their solid phase transformations during ball milling. Powder X-ray diffraction and modulated differential scanning calorimetry were used to characterize the samples after each milling time on their path to amorphization. Our results show that forms Ⅰ and III directly converted into an amorphous phase, while form Ⅱ first undergoes a polymorphic transition to form Ⅰ, and then gradually loses its crystallinity, finally reaching full amorphousness. During ball milling of forms Ⅰ and Ⅱ, the glass transition temperature (Tg) of the amorphous fraction of the milled material remains almost unchanged at 75 °C and 74 °C, respectively (whilst the amorphous content increases). In contrast, the Tg values of the amorphous fraction of milled form III increase with increasing milling times, from 63 °C to 71 °C, indicating an unexpected phenomenon of amorphous-to-amorphous transformation. The amorphous fraction of milled forms I and II samples presented a longer structural relaxation (i.e., lower molecular mobility) than the amorphous fraction of milled form III samples. Moreover, the structural relaxation time remained the same for the increasing amorphous fraction during milling of forms I and II. In contrast, the structural relaxation times were always shorter for the amorphous fraction of form III, but increased with increasing amorphous content during milling, confirming amorphous-to-amorphous transformation.

Exploring the effects of high pressure on hydrogen bonding in pharmaceutical cocrystals: A systematic study of pyridine dicarboxylic acid systems using synchrotron and neutron diffraction.

Pharmaceutical cocrystals use common robust hydrogen bonding synthons to create novel materials with different physicochemical properties. In this systematic study of a series of cocrystals, we explore the effect of high pressure on one of these commonly used motifs, the acid-pyridine motif, to assess the commonality of behaviour under extreme conditions. We have surveyed five pyridine dicarboxylic acid systems using both synchrotron and neutron diffraction methods to elucidate the changes in structure. We observe that the hydrogen bonding in these systems compress at a similar rate despite the changes to the molecular make-up of the solids and that on compression the changes in structure are indicative that the layers move along the major slip planes in the structure. We have observed two phase transitions to new forms of the pyrazine:malonic acid system, one for each stoichiometric ratio. This study demonstrates that the combination of two complementary diffraction approaches is key to understanding polymorphic behaviour at high pressure.

Composites made of Ginkgo biloba fibers and polylactic acid exhibit non-isothermal crystallization kinetics.

Polymer crystallization affects material microstructure and the final product quality, and the crystallization kinetics that govern this process are critical. In this study, alkali-treated Ginkgo biloba fibers (GFs) were melt blended with polylactic acid (PLA) to obtain GF/PLA blends. The non-isothermal crystallization kinetics of the GF/PLA composites were subsequently investigated using the Avrami, Jeziorny, Ozawa, and Liu-Mo methods, and the crystallization activation energies of the systems were calculated by Kissinger and Friedman models. The results showed that the GFs significantly promoted PLA crystallization, accelerated the crystallization rate, and shortened the crystallization time. The Avrami method showed some deviation from the linear relationship due to the effect of secondary crystallization, while the numeric value obtained by the Jeziorny method increased with the cooling rate. The Ozawa method could only be used in a very narrow range of temperatures, while the Liu-Mo method showed a more desirable fit. Crystallization activation energy calculations showed that the GFs promoted an increase in the crystallization capacity of the blend and a decrease in the effective potential barrier. This resulted in more selective biocomposites than pure PLA, offering greater applicability in domains including tissue engineering and 3D printing.

Ultrasound-assisted continuous crystallization of metastable polymorphic pharmaceutical in a slug-flow tubular crystallizer.

Metastable polymorphic pharmaceuticals have garnered significant attention in recent years due to their enhanced physicochemical properties, including solubility, bioavailability, and intellectual property considerations. However, the manufacturing of metastable form pharmaceuticals remains a formidable challenge. The stable preparation of metastable carvedilol (CVD) form Ⅱ crystals during CVD production is elusive, leading to substantial inconsistencies in product quality and regulatory compliance. In this study, we successfully prepared metastable CVD Form Ⅱ crystals using a continuous tubular crystallizer. Our findings demonstrate that the tubular crystallizer exhibits high efficiency and robustness for generating metastable crystal Form Ⅱ. We optimized the crystallization process by incorporating air bubble segments and employing ultrasonic irradiation strategies to overcome blockages and wall sticking issues encountered during operation. Ultimately, we developed an ultrasound-assisted continuous slug-flow tubular crystallization method and evaluated its performance. The results indicate that the CVD crystals produced through this process are resilient, sustainable, and uninterrupted products with promising potential for producing metastable polymorphic pharmaceuticals while effectively addressing encrustation problems associated with continuous tubular crystallization.