Protein crystallogenesis represents a key step in X-ray crystallography studies that employ co-crystallization and ligand soaking for investigating ligand binding to proteins. Co-crystallization is a method that enables the precise determination of binding positions, although it necessitates a significant degree of optimization. The utilization of microseeding can facilitate a reduction in sample requirements and accelerate the co-crystallization process. Ligand soaking is the preferred method due to its simplicity; however, it requires careful control of soaking conditions to ensure the successful integration of the ligands. This research protocol details the procedures for co-crystallization and soaking to achieve protein-ligand complex formation, which is essential for advancing drug discovery. Additionally, a simple protocol for demonstrating soaking for educational purposes is described.

• Klíčová slova
• advanced crystallization, co‐crystallization, crystal soaking, crystallization protocol, microseeding,
• MeSH
• konformace proteinů MeSH
• krystalizace metody   MeSH
• krystalografie rentgenová metody   MeSH
• ligandy MeSH
• proteiny * chemie   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ligandy MeSH
• proteiny * MeSH

The production of high-quality crystals is a key step in crystallography in general, but control of crystallization conditions is even more crucial in serial crystallography, which requires sets of crystals homogeneous in size and diffraction properties. This protocol describes the implementation of a simple and user-friendly microfluidic device that allows both the production of crystals by the counter-diffusion method and their in situ analysis by serial crystallography. As an illustration, the whole procedure is used to determine the crystal structure of three proteins from data collected at room temperature at a synchrotron radiation source.

• Klíčová slova
• CrystalChip, crystallization, microcrystals, microfluidics, serial crystallography,
• MeSH
• konformace proteinů MeSH
• krystalizace metody   přístrojové vybavení   MeSH
• krystalografie rentgenová metody   přístrojové vybavení   MeSH
• proteiny * chemie   MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny * MeSH

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.

• Klíčová slova
• CCP4, Coot, Cryoprotectant, Crystallization, Data collection, Data reduction, Indexing, Molecular replacement, PDB, Phasing, Protein homogeneity, Protein stability, Salt crystal, Scaling, Structure refinement, Twinning, XDS,
• MeSH
• konformace proteinů * MeSH
• krystalizace * metody   MeSH
• krystalografie rentgenová metody   MeSH
• molekulární modely MeSH
• proteiny * chemie   MeSH
• software MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• proteiny * MeSH

The present work reviews the liquid antisolvent crystallization (LASC) to prepare the nanoparticle of pharmaceutical compounds to enhance their solubility, dissolution rate, and bioavailability. The application of ultrasound and additives is discussed to prepare the particles with narrow size distribution. The use of ionic liquid as an alternative to conventional organic solvent is presented. Herbal compounds, also known for low aqueous solubility and limited clinical application, have been crystalized by LASC and discussed here. The particle characteristics such as particle size and particle size distribution are interpreted in terms of supersaturation, nucleation, and growth phenomena. To overcome the disadvantage of batch crystallization, the scientific literature on continuous flow reactors is also reviewed. LASC in a microfluidic device is emerging as a promising technique. The different design of the microfluidic device and their application in LASC are discussed. The combination of the LASC technique with traditional techniques such as high-pressure homogenization and spray drying is presented. A comparison of product characteristics prepared by LASC and the supercritical CO2 antisolvent method is discussed to show that LASC is an attractive and inexpensive alternative for nanoparticle preparation. One of the major strengths of this paper is a discussion on less-explored applications of LASC in pharmaceutical research to attract the attention of future researchers.

• Klíčová slova
• Antisolvent, Bioavailability, Crystallization, Microfluidic, Nanoparticle,
• MeSH
• farmaceutická technologie MeSH
• krystalizace metody   MeSH
• léčivé přípravky MeSH
• nanočástice * chemie   MeSH
• rozpouštědla chemie   MeSH
• rozpustnost MeSH
• velikost částic MeSH
• voda * MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• léčivé přípravky MeSH
• rozpouštědla MeSH
• voda * MeSH

Many efforts have been recently devoted to the design and investigation of multicomponent pharmaceutical solids, such as salts and cocrystals. The experimental distinction between these solid forms is often challenging. Here, we show that the transformation of a salt into a cocrystal with a short hydrogen bond does not occur as a sharp phase transition but rather a smooth shift of the positional probability of the hydrogen atoms. A combination of solid-state NMR spectroscopy, X-ray diffraction, and diffuse reflectance measurements with density functional theory calculations that include nuclear quantum effects (NQEs) provides evidence of temperature-induced hydrogen atom shift in cocrystals with short hydrogen bonds. We demonstrate that for the predictions of the salt/cocrystal solid forms with short H-bonds, the computations have to include NQEs (particularly hydrogen nuclei delocalization) and temperature effects.

• MeSH
• difrakce rentgenového záření MeSH
• krystalizace metody   MeSH
• léčivé přípravky MeSH
• vodík * MeSH
• vodíková vazba MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• léčivé přípravky MeSH
• vodík * MeSH

Seven inorganic salts containing N-phenylbiguanide as a prospective organic molecular carrier of nonlinear optical properties were prepared and studied within our research of novel hydrogen-bonded materials for nonlinear optics (NLO). All seven salts, namely N-phenylbiguanidium(1+) nitrate (C2/c), N-phenylbiguanidium(1+) perchlorate (P-1), N-phenylbiguanidium(1+) hydrogen carbonate (P21/c), bis(N-phenylbiguanidium(1+)) sulfate (C2), bis(N-phenylbiguanidium(1+)) hydrogen phosphate sesquihydrate (P-1), bis(N-phenylbiguanidium(1+)) phosphite (P21), and bis(N-phenylbiguanidium(1+)) phosphite dihydrate (P21/n), were characterised by X-ray diffraction (powder and single-crystal X-ray diffraction) and by vibrational spectroscopy (FTIR and Raman). Two salts with non-centrosymmetric crystal structures-bis(N-phenylbiguanidium(1+)) sulfate and bis(N-phenylbiguanidium(1+)) phosphite-were further studied to examine their linear and nonlinear optical properties using experimental and computational methods. As a highly SHG-efficient and phase-matchable material transparent down to 320 nm and thermally stable to 483 K, bis(N-phenylbiguanidium(1+)) sulfate is a promising novel candidate for NLO.

• Klíčová slova
• N-phenylbiguanidium(1+) cation, crystal structure, second harmonic generation, solid-state DFT, vibrational spectra,
• MeSH
• difrakce rentgenového záření metody   MeSH
• krystalizace metody   MeSH
• krystalografie rentgenová metody   MeSH
• optika a fotonika metody   MeSH
• Ramanova spektroskopie metody   MeSH
• soli chemie   MeSH
• spektroskopie infračervená s Fourierovou transformací metody   MeSH
• termogravimetrie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• soli MeSH

A new co-crystal of pharmaceutical active ingredient Apremilast was successfully designed in this work. The discovered co-crystal with benzoic acid significantly improves key properties like the dissolution and stability of an otherwise poorly soluble Apremilast. A crystallization process was developed, which includes efficient solvent selection and ternary phase diagram construction to minimize risks during scale up. To increase efficiency, we propose that both steps be combined into a single methodology based on solubility data. A suitable solvent for the co-crystallization process was selected and ternary phase diagrams were constructed using three different modifications of thermodynamic model of solid-liquid equilibria. Based on the obtained information, the co-crystallization process was scaled-up to 100 mL. This provides a feasible process to produce larger amounts of this promising pharmaceutical solid form of Apremilast necessary for further drug development.

• Klíčová slova
• Apremilast, Benzoic acid, Co-crystal, Crystallization, Solubility, Ternary phase diagram, Thermodynamic model,
• MeSH
• krystalizace metody   MeSH
• kyselina benzoová chemie   MeSH
• rozpouštědla chemie   MeSH
• rozpustnost účinky léků   MeSH
• termodynamika MeSH
• thalidomid analogy a deriváty   chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• apremilast MeSH Prohlížeč
• kyselina benzoová MeSH
• rozpouštědla MeSH
• thalidomid MeSH

We demonstrate the use of two nuclear-based analytical methods that can follow the modifications of microstructural arrangement of iron-based metallic glasses (MGs). Despite their amorphous nature, the identification of hyperfine interactions unveils faint structural modifications. For this purpose, we have employed two techniques that utilize nuclear resonance among nuclear levels of a stable 57Fe isotope, namely Mössbauer spectrometry and nuclear forward scattering (NFS) of synchrotron radiation. The effects of heat treatment upon (Fe2.85Co1)77Mo8Cu1B14 MG are discussed using the results of ex situ and in situ experiments, respectively. As both methods are sensitive to hyperfine interactions, information on structural arrangement as well as on magnetic microstructure is readily available. Mössbauer spectrometry performed ex situ describes how the structural arrangement and magnetic microstructure appears at room temperature after the annealing under certain conditions (temperature, time), and thus this technique inspects steady states. On the other hand, NFS data are recorded in situ during dynamically changing temperature and NFS examines transient states. The use of both techniques provides complementary information. In general, they can be applied to any suitable system in which it is important to know its steady state but also transient states.

• MeSH
• krystalizace metody   MeSH
• povrchové vlastnosti MeSH
• slitiny chemie   MeSH
• testování materiálů MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• slitiny MeSH

Human aldo-keto reductase 1C3 (AKR1C3) stereospecifically reduces steroids and prostaglandins and is involved in the biotransformation of xenobiotics. Its role in various cancers makes it a potential therapeutic target for the development of inhibitors. Recombinant AKR1C3 with a thrombin-cleavable N-terminal His6 tag was expressed from a pET-28(+) vector for structural studies of enzyme-inhibitor complexes. A modified in situ proteolysis approach was applied to specifically remove the His tag by thrombin cleavage during crystallization screening trials. This improved the morphology and diffraction quality of the crystals and allowed the acquisition of high-resolution diffraction data and structure solution. This approach may be generally applicable to other proteins expressed using the pET-28(+) vector.

• Klíčová slova
• 17β-hydroxysteroid dehydrogenase 5, His tags, aldo-keto reductase 1C3, diffraction-quality improvement, in situ proteolysis, pET-28(+),
• MeSH
• difrakce rentgenového záření metody   MeSH
• histidin * genetika   MeSH
• krystalizace metody   MeSH
• krystalografie rentgenová metody   MeSH
• lidé MeSH
• protein AKR1C3 chemie   genetika   metabolismus   MeSH
• proteolýza MeSH
• sekvence aminokyselin MeSH
• trombin metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AKR1C3 protein, human MeSH Prohlížeč
• histidin * MeSH
• protein AKR1C3 MeSH
• trombin MeSH

A sustainable way to recover phosphorus (P) in swine wastewater involves a preliminary step of P dissolution followed by the separation of particulate organic matter (OM). The next two steps are firstly the precipitation of struvite crystals done by adding a crystallization reagent (magnesia) and secondly the filtration of the crystals. To develop the process successfully at an industrial scale, the control of the mechanisms of precipitation is the key point in order to obtain high value-added products, that is, big struvite crystals easy to harvest and handle. Experiments with process parameters optimized previously in a synthetic swine wastewater were performed on real swine wastewater to assess the role of the OM on struvite crystallization. After 24 h, with a pH increase to 6.8 only, 90% of the initial P was precipitated and 60% was precipitated as struvite. 80% of the solid recovered was in the fraction > 100 µm. The other forms recovered were brushite, amorphous calcium phosphate, NaCl, KCl and OM. The influence of OM on struvite precipitation in acidified swine wastewater was negative on the reaction kinetics but positive on the size of the struvite crystals. The presence of colloidal particles increased the size of the struvite crystals but slowed down the kinetics due to the viscosity induced by the repulsive force of the colloids. The maximum size of single struvite crystals (200 µm) was observed with the presence of particulate OM.

• Klíčová slova
• Struvite, organic matter, phosphorus recovery, pig manure, swine wastewater,
• MeSH
• fosfáty chemie   MeSH
• fosfor chemie   MeSH
• hnůj MeSH
• koloidy chemie   MeSH
• koncentrace vodíkových iontů MeSH
• krystalizace metody   MeSH
• odpad tekutý - odstraňování MeSH
• odpadní voda chemie   MeSH
• prasata MeSH
• sloučeniny hořčíku chemie   MeSH
• struvit MeSH
• velikost částic MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfáty MeSH
• fosfor MeSH
• hnůj MeSH
• koloidy MeSH
• odpadní voda MeSH
• sloučeniny hořčíku MeSH
• struvit MeSH