The crystallization operation is widely used in pharmaceutical production processes. Crystallization is one of unit operation which deals with crystallization phenomena. Crystal qualities have a huge impact on downstream processes and hence affect the purity and cost of the pharmaceuticals. So it is important to control crystal qualities. The driving force of crystallization is supersaturation. Therefore design strategy of supersaturation is necessary to control the crystal qualities. The crystal qualities strongly depend on the trajectory of solution concentration because crystallization phenomena proceed in non-equilibrium process. In order to understand the relationships between operation condition and product specification, the consideration of both equilibrium and kinetics of crystallization by using phase diagram is necessary. In this paper, the operation design method and operation strategy are introduced to control crystal qualities in crystallization by understanding the influence of a dynamic change of the solution composition on the crystal qualities.
Protein crystals are generally wet and fragile, thus easily suffer from dehydration and temperature change. To preserve the crystals from such sudden physicochemical conditional changes, we developed a crystal mounting method, humid air and glue coating (HAG) method. This method works well to maintain most protein crystals under both cryogenic and room temperature conditions. Room-temperature measurement has recently advanced in time-resolved analysis by serial femtosecond crystallography (SFX) method using X-ray free electron laser (XFEL) facilities, and emerges dynamical properties of proteins, leading to its recurrence even in synchrotron experiments. Here we show the brief guidance to the method and its application, and discuss the perspective of room-temperature structural analysis in macromolecular crystallography.
We describe new methods for selective crystallization of the metastable phase of pharmaceutical compounds using a slow cooling method, polymer-induced heteronucleation (PIHn) and solution mediated transformation. New crystal seeding methods were also introduced, thus it became possible to control the supersaturations. These technology enabled us to improve quality of the metastable phase．Appropriate control of the form II crystal growth rate via slow cooling or solution mediated transformation suppressed the defect formation and improved the crystallinity of form II. These polymorphic control technologies are applicable to other materials, thus they have possibility to be practical applications in the future.
The sugar chains operate as tags for intracellular quality control of glycoproteins, ensuring their appropriate folding and trafficking in cells. Especially, in the endoplasmic reticulum, the folding states of newly synthesized proteins are recognized through the presence or absence of only a single terminal glucose residue. These key processes involving glucose-trimming and attachment are executed by actions of glucosidase II and UDP-glucose/glycoprotein glucosyltransferase (UGGT), respectively. Recently, the eukaryotic multi-domain, large enzymes that are generally difficult to crystallize, were successfully crystallized by utilizing thermophilic fungi as the source organisms. This review summarizes a recently emerging evidence regarding structural basis of the functional mechanisms of these key enzymes, as provided by X-ray crystallography in conjunction with a series of biophysical techniques, including small-angle X-ray scattering, high-speed atomic force microscopy and electron microscopy, which are capable of providing dynamic views of these enzymes in solution.
DNA damage tolerance (DDT) is a cell function to release replication blockage caused by DNA damage and to continue DNA synthesis even in the presence of DNA damage. DDT includes two pathways, translesion and template-switched DNA syntheses. Helicase-like transcription factor (HLTF) is a central protein in the template-switched DNA synthesis pathway, in which one newly synthesized strand is utilized as an undamaged template for replication. HLTF consists of three domains, N-terminal DNA binding, DNA helicase, and ubiquitin ligase domains. The N-terminal DNA binding domain is termed HIRAN domain. Here, we outline the crystal structure of HIRAN domain bound to DNA and its functional implication in template-switched DNA synthesis. Furthermore, we describe that artifacts in crystallization gave fruitful results in our structural study of HIRAN domain.