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

Nepenthesins are aspartic proteases secreted by carnivorous pitcher plants of the genus Nepenthes. They significantly differ in sequence from other plant aspartic proteases. This difference, which provides more cysteine residues in the structure of nepenthesins, may contribute to their unique stability profile. Recombinantly produced nepenthesin 1 (rNep1) from N. gracilis in complex with pepstatin A was crystallized under two different crystallization conditions using a newly formulated low-pH crystallization screen. The diffraction data were processed to 2.9 and 2.8 Å resolution, respectively. The crystals belonged to space group P212121, with unit-cell parameters a = 86.63, b = 95.90, c = 105.40 Å, α = β = γ = 90° and a = 86.28, b = 97.22, c = 103.78 Å, α = β = γ = 90°, respectively. Matthews coefficient and solvent-content calculations suggest the presence of two molecules of rNep1 in the asymmetric unit. Here, the details of the crystallization experiment and analysis of the X-ray data are reported.

• Klíčová slova
• Nepenthes gracilis, aspartic proteases, low-pH crystallization screen, nepenthesins,
• MeSH
• aspartátové endopeptidasy chemie   MeSH
• koncentrace vodíkových iontů MeSH
• krystalizace MeSH
• krystalografie rentgenová MeSH
• Magnoliopsida enzymologie   MeSH
• pepstatiny chemie   MeSH
• rostlinné proteiny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aspartátové endopeptidasy MeSH
• pepstatin MeSH Prohlížeč
• pepstatiny MeSH
• rostlinné 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

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

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

The self-plasticization, i.e., the increase in the polymer chains' mobility by including its monomer, has a major impact on a polymer's structural, thermal, and mechanical properties. In this study, differential scanning calorimetry (DSC), optical and Raman microscopies, thermo-mechanical analysis (TMA), size exclusion chromatography equipped with a multi-angle light scattering detector (SEC-MALS), and X-ray diffraction analysis (XRD) are used to investigate the effect of thermally induced self-plasticization of poly-(p-dioxanone), PDX, on the crystal growths from the amorphous and molten states. Significant changes in the crystallization behavior and mechanical properties of PDX are found only for samples self-plasticized at the depolymerization temperature (Td) above 150 °C. The intense self-plasticization leads to the decrease of the crystallization temperature, increase of the crystal growth rapidity, disappearance of the distinct α→α' polymorphic transition, reduction of the overall melting temperature, and segregation of the redundant monomer. Although the morphology of the crystalline phase has a major impact on the mechanical properties of PDX, the self-plasticization itself does not seem to result in any major changes in the magnitude, localization, or morphology of formed crystallites (these are primarily driven by the temperature of crystal growth). The manifestation of the variable activation energy concept is discussed for the present crystallization data.

• Klíčová slova
• crystallization, depolymerization, differential scanning calorimetry, polydioxanone, self‐plasticization,
• MeSH
• diferenciální skenovací kalorimetrie MeSH
• difrakce rentgenového záření MeSH
• dioxany * chemie   MeSH
• krystalizace * MeSH
• nízká teplota MeSH
• polymerizace * MeSH
• polymery * chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dioxany * MeSH
• poly-4-dioxan-2-one MeSH Prohlížeč
• polymery * MeSH

The newly discovered di-haem cytochrome c4 from the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina is the first cytochrome c4 to be crystallized from an anaerobic organism. It was crystallized using the addition of metal-ion salts to the standard vapour-diffusion method. Coloured well shaped three-dimensional crystals with dimensions of approximately 0.6 x 0.05 x 0.02 mm grew within 3-4 d at pH 5 and diffracted to 1.72 angstroms without radiation damage. Cytochrome c4 crystallized in space group P4(1)2(1)2 as a primitive tetragonal system with unit-cell parameters a = b = 75.29, c = 37.12 angstroms, alpha = beta = gamma = 90 degrees.

• MeSH
• bakteriální proteiny chemie   izolace a purifikace   MeSH
• cytochromy skupiny c chemie   izolace a purifikace   MeSH
• difrakce rentgenového záření MeSH
• krystalizace MeSH
• Thiocapsa enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• cytochrome C4 MeSH Prohlížeč
• cytochromy skupiny c MeSH

The molecules adhering temporarily on the surface of protein molecules change the propensity of protein molecules to deposit on the crystal surface in a definite position and orientation. The concepts of competitive adhesion modes and protein surface shielding agents acting on the surface of molecules in a non-equilibrium process of protein crystallization provide a useful platform for the control of crystallization. The desirable goal, i.e. a transient preference of a single dominating adhesion mode between protein molecules during crystallization, leads to uniform deposition of proteins in a crystal. This condition is the most important factor for diffraction quality and thus also for the accuracy of protein structure determination. The presented hypothesis is a generalization of the experimentally well proven behaviour of hydrophilic polymers on the surface of protein molecules of other compounds.

• MeSH
• difrakce rentgenového záření MeSH
• hydrofobní a hydrofilní interakce MeSH
• krystalizace * metody   MeSH
• polyethylenglykoly chemie   MeSH
• povrchové vlastnosti MeSH
• proteiny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• polyethylenglykoly MeSH
• proteiny MeSH

Time-resolved structural investigations of crystallization of water in lipid/protein/salt mesophases at cryogenic temperatures are significant for comprehension of ice nanocrystal nucleation kinetics in lipid membranous systems and can lead to a better understanding of how to experimentally retard the ice formation that obstructs the protein crystal structure determination. Here, we present a time-resolved synchrotron microfocus X-ray diffraction (TR-XRD) study based on ∼40,000 frames that revealed the dynamics of water-to-ice crystallization in a lipid/protein/salt mesophase subjected to cryostream cooling at 100 K. The monoolein/hemoglobin/salt/water system was chosen as a model composition related to protein-loaded lipid cubic phases (LCP) broadly used for the crystallization of proteins. Under confinement in the nanoscale geometry, metastable short-living cubic ice (Ic) rapidly crystallized well before the formation of hexagonal ice (Ih). The detected early nanocrystalline states of water-to-ice transformation in multicomponent systems are relevant to a broad spectrum of technologies and understanding of natural phenomena, including crystallization, physics of water nanoconfinement, and rational design of anti-freezing and cryopreservation systems.

• Klíčová slova
• Crystallization kinetics, Cubic ice, In situ microfocus beam X-ray diffraction, Lipid cubic phase, Monoolein/hemoglobin/water assembly, Nanoconfinement, Structural dynamics,
• MeSH
• krystalizace MeSH
• led * MeSH
• nízká teplota MeSH
• voda * chemie   MeSH
• změna skupenství MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• led * MeSH
• voda * MeSH

The endonuclease TBN1 from Solanum lycopersicum (tomato) was expressed in Nicotiana benthamiana leaves and purified with suitable quality and in suitable quantities for crystallization experiments. Two crystal forms (orthorhombic and rhombohedral) were obtained and X-ray diffraction experiments were performed. The presence of natively bound Zn2+ ions was confirmed by X-ray fluorescence and by an absorption-edge scan. X-ray diffraction data were collected from the orthorhombic (resolution of 5.2 Å) and rhombohedral (best resolution of 3.2 Å) crystal forms. SAD, MAD and MR methods were applied for solution of the phase problem, with partial success. TBN1 contains three Zn2+ ions in a similar spatial arrangement to that observed in nuclease P1 from Penicillium citrinum.

• MeSH
• deoxyribonukleasy chemie   genetika   MeSH
• ionty chemie   MeSH
• konformace proteinů MeSH
• krystalizace MeSH
• krystalografie rentgenová MeSH
• molekulární sekvence - údaje MeSH
• rekombinantní proteiny chemie   genetika   MeSH
• rostlinné proteiny chemie   genetika   MeSH
• Solanum lycopersicum chemie   genetika   MeSH
• zinek chemie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• deoxyribonukleasy MeSH
• ionty MeSH
• rekombinantní proteiny MeSH
• rostlinné proteiny MeSH
• zinek MeSH