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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. Co‐crystallization crystallizes a protein with its ligand, resulting in protein–ligand complex crystals. In contrast, soaking introduces a ligand into preformed protein crystals, allowing it to bind. Both methods produce crystals for X‐ray diffraction, which generates diffraction patterns that are analyzed to determine the three‐dimensional structure of the complex. This process uncovers key interactions critical to understanding the protein's biological functions.

There is growing interest in using antibodies as auxiliary tools to crystallize proteins. Here we describe a general protocol for the generation of Nanobodies to be used as crystallization chaperones for the structural investigation of diverse conformational states of flexible (membrane) proteins and complexes thereof. Our technology has a competitive advantage over other recombinant crystallization chaperones in that we fully exploit the natural humoral response against native antigens. Accordingly, we provide detailed protocols for the immunization with native proteins and for the selection by phage display of in vivo –matured Nanobodies that bind conformational epitopes of functional proteins. Three representative examples illustrate that the outlined procedures are robust, making it possible to solve by Nanobody-assisted X-ray crystallography in a time span of 6–12 months.

A detailed protocol for crystallizing membrane proteins that makes use of lipidic mesophases is described. This has variously been referred to as the lipid cubic phase or in meso method. The method has been shown to be quite general in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and α-helical and β-barrel proteins. Its most recent successes are the human-engineered β 2 -adrenergic and adenosine A 2A G protein–coupled receptors. Protocols are provided for preparing and characterizing the lipidic mesophase, for reconstituting the protein into the monoolein-based mesophase, for functional assay of the protein in the mesophase and for setting up crystallizations in manual mode. Methods for harvesting microcrystals are also described. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about 1 h.

Magnetic nanoparticles are increasingly used in medical applications, including cancer treatment by magnetic hyperthermia. This protocol describes a solvothermal-based process to prepare, at the gram scale, ferrite nanoparticles with well-defined shape, i.e., nanocubes, nanostars and other faceted nanoparticles, and with fine control of structural/magnetic properties to achieve point-of-reference magnetic hyperthermia performance. This straightforward method comprises simple steps: (i) making a homogeneous alcoholic solution of a surfactant and an alkyl amine; (ii) adding an organometallic metal precursor together with an aldehyde molecule, which acts as the key shape directing agent; and (iii) reacting the mixture in an autoclave for solvothermal crystallization. The shape of the ferrite nanoparticles can be controlled by the structure of the aldehyde ligand. Benzaldehyde and its aromatic derivatives favor the formation of cubic ferrite nanoparticles while aliphatic aldehydes result in spherical nanoparticles. The replacement of the primary amine, used in the nanocubes synthesis, with a secondary/tertiary amine results in nanoparticles with star-like shape. The well-defined control in terms of shape, narrow size distribution (below 5%), compositional tuning and crystallinity guarantees the preparation, at the gram scale, of nanocubes/star-like nanoparticles that possess, under magnetic field conditions of clinical use, specific adsorption rates comparable to or even superior to those obtained through thermal decomposition methods, which are typically prepared at the milligram scale. Here, gram-scale nanoparticle products with benchmark features for magnetic hyperthermia applications can be prepared in ~10 h with an average level of expertise in chemistry. This protocol describes a solvothermal-based process to prepare gram-scale ferrite nanoparticles with well-defined shapes (nanocubes, nanostars, faceted and spherical) having heating properties appealing for clinical magnetic hyperthermia treatments.

Covalent organic frameworks (COFs) are crystalline porous polymers constructed from organic building blocks into ordered two- or three-dimensional networks through dynamic covalent bonds. Attributed to their high porosity, well-defined structure, tailored functionality and excellent chemical stability, COFs have been considered ideal sorbents for various separation applications. The synthesis of COFs mainly employs the solvothermal method, which usually requires organic solvents in sealed Pyrex tubes, resulting in unscalable powdery products and environmental pollution that seriously limits their practical applications. Herein, our protocol focuses on an emerging synthesis method for COFs based on organic flux synthesis without adding solvents. The generality of this synthesis protocol has been applied in preparing various types of COFs, including olefin-linked, imide-linked, Schiff-based COFs on both gram and kilogram scales. Furthermore, organic flux synthesis avoids the disadvantages of solvothermal synthesis and enhances the crystallization and porosity of COFs. Typically, COF synthesis takes 3–5 d to complete, and subsequent washing procedures leading to pure COFs need 1 d. The procedure for kilogram-scale production of COFs with commercially available monomers is also provided. The resulting COFs are suitable for separation applications, particularly as adsorbent materials for industrial gas separation and water treatment applications. The protocol is suited for users with prior expertise in the synthesis of inorganic materials and porous organic materials. Key points This protocol describes a flux synthesis approach for two-dimensional covalent organic frameworks (COFs). Compared with other approaches, the method described here does not use solvents, making it environmentally friendly, and is scalable up to the kilogram scale. In addition, high-quality COF monoliths can be generated rather than powdery products, and COFs prepared by this protocol usually possess higher crystallinity and BET surface area than those prepared by traditional solvothermal methods. This protocol describes a flux synthesis approach for two-dimensional covalent organic frameworks. Compared with other approaches, this method does not use solvents, making it environmentally friendly, and is scalable up to the kilogram scale.

Crystallization of recombinant proteins in living cells is an emerging approach complementing conventional crystallization techniques. Homogeneous microcrystals well suited for serial diffraction experiments at X‐ray free‐electron lasers and synchrotron sources can be produced in a quasi‐native environment, without the need for target protein purification. Several protein structures have already been solved; however, exploiting the full potential of this approach requires a systematic and versatile screening strategy for intracellular crystal growth. Recently, we published InCellCryst, a streamlined pipeline for producing microcrystals within living insect cells. Here, we present the detailed protocol, including optimized target gene expression using a baculovirus vector system, crystal formation, detection, and serial X‐ray diffraction directly in the cells. The specific environment within the different cellular compartments acts as a screening parameter to maximize the probability of crystal growth. If successful, diffraction data can be collected 24 days after the start of target gene cloning. This research protocol provides a step‐by‐step guide for applying the InCellCryst pipeline for intracellular protein crystallization. After gene cloning and generation of recombinant baculoviruses, High Five insect cells are infected for target protein crystallization. If detection is successful, the crystal‐containing cells are mounted for serial X‐ray diffraction data collection directly in the cells, followed by data processing and structure elucidation.

A protocol for the preparation of crystals for microcrystal electron diffraction provides X-ray crystallographers and cryo-electron microscopists access to a method that facilitates the determination of molecular structures from submicrometer crystals at atomic resolution.

Kinases are a widely targeted enzyme class in cancer chemotherapy. Several clinically used kinase inhibitors also inhibit bromodomains, epigenetic ‘readers’ of acetylated lysine residues, suggesting that kinase-bromodomain polypharmacology may offer benefits in therapeutic settings. Concomitant inhibition of multiple cancer-driving kinases is an established strategy to improve the durability of clinical responses to targeted therapies. The difficulty of discovering kinase inhibitors with an appropriate multitarget profile has, however, necessitated the application of combination therapies, which can pose major clinical development challenges. Epigenetic reader domains of the bromodomain family have recently emerged as new targets for cancer therapy. Here we report that several clinical kinase inhibitors also inhibit bromodomains with therapeutically relevant potencies and are best classified as dual kinase-bromodomain inhibitors. Nanomolar activity on BRD4 by BI-2536 and TG-101348, which are clinical PLK1 and JAK2-FLT3 kinase inhibitors, respectively, is particularly noteworthy as these combinations of activities on independent oncogenic pathways exemplify a new strategy for rational single-agent polypharmacological targeting. Furthermore, structure-activity relationships and co-crystal structures identify design features that enable a general platform for the rational design of dual kinase-bromodomain inhibitors.

Thermoplastic composite pipes (TCP) consist of three distinct layers—liner, reinforcement, and coating—offering superior advantages over traditional industrial pipes, including flexibility, lightweight construction, and corrosion resistance. This study systematically characterises the thermal properties of TCP layers and their compositions using a multi-method approach. Thermal analysis was conducted through differential scanning calorimetry (DSC) for isothermal and non-isothermal crystallisation, thermogravimetric analysis (TGA) for thermal stability, and Fourier transform infrared spectroscopy (FTIR) for material identification. FTIR confirmed polyethylene as the primary component of TCP, with compositional variations across the layers. TGA results indicated that thermal degradation begins at approximately 200 °C, with complete decomposition at 500 °C. DSC analysis revealed a double melting peak, prompting further investigation into its mechanisms. On-isothermal crystallisation kinetics, analysed at cooling rates of 10 °C/min and 50 °C/min, revealed an anisotropic crystalline growth pattern. Although nucleation occurs uniformly, the subsequent three-dimensional crystalline growth is governed more by the degree of supercooling than by the crystallography of the glass fibres. This underscores the importance of precisely controlling the cooling rate during manufacturing to optimise the anisotropic properties of the reinforced layer. This study also demonstrates the value of FTIR, TGA, and DSC techniques in characterising the thermo-physical behaviour of TCP, offering critical insights into thermal expansion, shrinkage phenomena, and overall material stability. Given the limited body of research on this specific TCP formulation, the findings presented here lay a foundation for both quality enhancement and process optimisation. Moreover, the paper offers a distinctive perspective on the dynamic behaviour, thermal expansion, and long-term performance of TCP in demanding oil and gas environments.

A Protocol for obtaining high-quality protein crystals that involves addition to the protein solution of solid or semi-liquid nucleants that provide the experimenter the means to control crystal growth. Solving the structure of proteins is pivotal to achieving success in rational drug design and in other biotechnological endeavors. The most powerful method for determining the structure of proteins is X-ray crystallography, which relies on the availability of high-quality crystals. However, obtaining such crystals is a major hurdle. Nucleation is the crucial prerequisite step, which requires overcoming an energy barrier. The presence in a protein solution of a nucleant, a solid or a semiliquid substance that facilitates overcoming that barrier allows crystals to grow under ideal conditions, paving the way for the formation of high-quality crystals. The use of nucleants provides a unique means for optimizing the diffraction quality of crystals, as well as for discovering new crystallization conditions. We present a protocol for controlling the nucleation of protein crystals that is applicable to a wide variety of nucleation-inducing substances. Setting up crystallization trials using these nucleating agents takes an additional few seconds compared with conventional setup, and it can accelerate crystallization, which typically takes several days to months.