-
Journal of Visualized Experiments : JoVE Jan 2011Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of...
Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography. To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999). Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop. Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.
Topics: Crystallization; Crystallography, X-Ray; Diffusion; Proteins
PubMed: 21304455
DOI: 10.3791/2285 -
Acta Crystallographica. Section F,... May 2022The CENP-SX (MHF) complex is a conserved histone-fold protein complex that is involved in chromosome segregation and DNA repair. It can bind to DNA on its own as well as...
The CENP-SX (MHF) complex is a conserved histone-fold protein complex that is involved in chromosome segregation and DNA repair. It can bind to DNA on its own as well as in complex with other proteins such as CENP-TW and FANCM to recognize specific substrates. CENP-SX binds nonspecifically to dsDNA, similar to other histone-fold proteins. Several low-resolution structures of CENP-SX in complex with DNA are known, but a high-resolution structure is still lacking. The DNA-binding properties of CENP-SX and FANCM-CENP-SX complexes with various lengths of dsDNA were compared and the band-shift patterns and migration positions were found to differ. To confirm the DNA-binding properties in detail, CENP-SX-DNA and FANCM-CENP-SX-DNA complexes were crystallized. Analysis of the crystals revealed that they all contained the CENP-SX-DNA complex, irrespective of the complex that was used in crystallization. Detailed diffraction data analyses revealed that there were two types of crystal with different space groups, P2 and C2, where the volume of the P2 asymmetric unit is twice as large as that of the C2 asymmetric unit. Analysis of the self-rotation function revealed the presence of twofold and fourfold symmetry in both crystals. This suggests that there may be multiple molecules of CENP-SX and DNA within the asymmetric unit with respective symmetry. Structure determination of the present crystals should reveal details of the DNA-binding properties of CENP-SX.
Topics: Crystallization; Crystallography, X-Ray; DNA; DNA-Binding Proteins; Histones
PubMed: 35506764
DOI: 10.1107/S2053230X22003843 -
Archives of Biochemistry and Biophysics Jul 2016Crystallization is a key step in macromolecular structure determination by crystallography. While a robust theoretical treatment of the process is available, due to the... (Review)
Review
Crystallization is a key step in macromolecular structure determination by crystallography. While a robust theoretical treatment of the process is available, due to the complexity of the system, the experimental process is still largely one of trial and error. In this article, efforts in the field are discussed together with a theoretical underpinning using a solubility phase diagram. Prior knowledge has been used to develop tools that computationally predict the crystallization outcome and define mutational approaches that enhance the likelihood of crystallization. For the most part these tools are based on binary outcomes (crystal or no crystal), and the full information contained in an assembly of crystallization screening experiments is lost. The potential of this additional information is illustrated by examples where new biological knowledge can be obtained and where a target can be sub-categorized to predict which class of reagents provides the crystallization driving force. Computational analysis of crystallization requires complete and correctly formatted data. While massive crystallization screening efforts are under way, the data available from many of these studies are sparse. The potential for this data and the steps needed to realize this potential are discussed.
Topics: Computer Simulation; Crystallization; Crystallography; Models, Molecular; Protein Conformation; Proteins
PubMed: 26792536
DOI: 10.1016/j.abb.2016.01.004 -
Ultrasonics Sonochemistry Nov 2014The application of ultrasound to crystallization (i.e., sonocrystallization) can dramatically affect the properties of the crystalline products. Sonocrystallization... (Review)
Review
The application of ultrasound to crystallization (i.e., sonocrystallization) can dramatically affect the properties of the crystalline products. Sonocrystallization induces rapid nucleation that generally yields smaller crystals of a more narrow size distribution compared to quiescent crystallizations. The mechanism by which ultrasound induces nucleation remains unclear although reports show the potential contributions of shockwaves and increases in heterogeneous nucleation. In addition, the fragmentation of molecular crystals during ultrasonic irradiation is an emerging aspect of sonocrystallization and nucleation. Decoupling experiments were performed to confirm that interactions between shockwaves and crystals are the main contributors to crystal breakage. In this review, we build upon previous studies and emphasize the effects of ultrasound on the crystallization of organic molecules. Recent work on the applications of sonocrystallized materials in pharmaceutics and materials science are also discussed.
Topics: Crystallization; Time Factors; Ultrasonics
PubMed: 24636362
DOI: 10.1016/j.ultsonch.2014.02.005 -
Methods in Molecular Biology (Clifton,... 2021Here, we present a strategy to identify microcrystals from initial protein crystallization screen experiments and to optimize diffraction quality of those crystals using...
Here, we present a strategy to identify microcrystals from initial protein crystallization screen experiments and to optimize diffraction quality of those crystals using negative stain transmission electron microscopy (TEM) as a guiding technique. The use of negative stain TEM allows visualization along the process and thus enables optimization of crystal diffraction by monitoring the lattice quality of crystallization conditions. Nanocrystals bearing perfect lattices are seeded and can be used for MicroED as well as growing larger crystals for X-ray and free electron laser (FEL) data collection.
Topics: Cryoelectron Microscopy; Crystallization; Microscopy, Electron, Transmission; Nanoparticles; Protein Conformation
PubMed: 33368010
DOI: 10.1007/978-1-0716-0966-8_14 -
Journal of Dairy Science Feb 2021Scientific interest in cheese crystals extends back more than a century. However, starting around the 1970s, industry interest, and interest on the part of cheese... (Review)
Review
Scientific interest in cheese crystals extends back more than a century. However, starting around the 1970s, industry interest, and interest on the part of cheese scientists, grew dramatically as changes in cheesemaking technology and market changes caused the presence of crystals in the marketplace to increase; advanced analytical capabilities enabled new crystalline species to be identified, their origins and causative factors to be elucidated, and their contributions to cheese texture to be better understood. It is now evident that a host of organic- and inorganic-based crystals occur in natural cheeses. Some crystals form preferentially at the surface of rindless or rinded cheeses, others in the irregular openings or spherical eyes that occur within the body of some cheeses, and still others embedded within the cheese matrix. It is also evident that crystals may profoundly influence cheese texture, both as a direct consequence of their abundance, size, shape, and hardness, and as an indirect result of cascading physiochemical events initiated by crystal formation. Consumer response to increased incidence of crystals in the marketplace has been mixed. On the one hand, surface crystals of calcium lactate pentahydrate on Cheddar cheese came to be viewed quite negatively in some markets, often being mistaken for mold growth and spoilage. This triggered industry concern and led to considerable research to determine the underlying causes and to develop strategies to limit or prevent calcium lactate pentahydrate formation. At the same time, other forms of crystallization increasingly came to be viewed as positive features in the growing market dedicated to artisanal and traditional cheeses, giving rise to a bifurcated consumer response to cheese crystals that is evident today. Traditional artisanal cheesemakers perhaps have the most to gain from advances in cheese-crystal research. Traditional artisanal cheeses rely heavily on stories that are weaved around their identity to create uniqueness and add value. A challenge and opportunity for these cheesemakers in the United States and globally will be to translate the fascinating science of their cheese crystals into engaging narratives that capture the imagination, add value to their cheese, and enhance the enjoyment of their cheese by consumers.
Topics: Animals; Calcium Compounds; Cheese; Chemical Phenomena; Crystallization; Food Handling; Food Technology; Lactates; Sensation
PubMed: 33309343
DOI: 10.3168/jds.2020-19119 -
Molecules (Basel, Switzerland) Nov 2022Tafamidis, chemical formula CHClNO, is a drug used to delay disease progression in adults suffering from transthyretin amyloidosis, and is marketed worldwide under...
Tafamidis, chemical formula CHClNO, is a drug used to delay disease progression in adults suffering from transthyretin amyloidosis, and is marketed worldwide under different tradenames as a free acid or in the form of its meglumine salt. The free acid (CAS no. 594839-88-0) is reported to crystallize as distinct (polymorphic) crystal forms, the thermal stability and structural features of which remained thus far undisclosed. In this paper, we present-by selectively isolating highly pure batches of Tafamidis Form 1 and Tafamidis Form 4-the full characterization of these solids, in terms of crystal structures (determined using state-of-the-art structural powder diffraction methods) and spectroscopic and thermal properties. Beyond conventional thermogravimetric and calorimetric analyses, variable-temperature X-ray diffraction was employed to measure the highly anisotropic response of these (poly)crystalline materials to thermal stimuli and enabled the determination of the linear and volumetric thermal expansion coefficients and of the related indicatrix. Both crystal phases are monoclinic and contain substantially flat and π-π stacked Tafamidis molecules, arranged as centrosymmetric dimers by strong O-H···O bonds; weaker C-H···N contacts give rise, in both polymorphs, to infinite ribbons, which guarantee the substantial stiffness of the crystals in the direction of their elongation. Complete knowledge of the structural models will foster the usage of full-pattern quantitative phase analyses of Tafamidis in drug and polymorphic mixtures, an important aspect in both the forensic and the industrial sectors.
Topics: Crystallization; Powder Diffraction; X-Ray Diffraction
PubMed: 36364244
DOI: 10.3390/molecules27217411 -
Current Allergy and Asthma Reports Jun 2019Charcot-Leyden crystals (CLCs), slender bipyramidal hexagonal crystals, were first described by Jean-Martin Charcot in 1853, predating Paul Ehrlich's "discovery" of... (Review)
Review
PURPOSE OF REVIEW
Charcot-Leyden crystals (CLCs), slender bipyramidal hexagonal crystals, were first described by Jean-Martin Charcot in 1853, predating Paul Ehrlich's "discovery" of eosinophils by 26 years. To date, CLCs are known as a classical hallmark of eosinophilic inflammation. CLC protein expresses palmitate cleaving lysophospholipase activity and is a member of the family of S-type lectins, galectin-10. We summarize current knowledge regarding the pathological observations of CLCs and their mechanism of generation focusing on eosinophil cell death.
RECENT FINDINGS
The presence of CLCs in vivo has been consistently associated with lytic eosinophils. Recent evidence revealed that cytolysis represents the occurrence of extracellular trap cell death (ETosis), an active non-apoptotic cell death process releasing filamentous chromatin structure. Galectin-10 is a predominant protein present within the cytoplasm of eosinophils but not stored in secretory granules. Activated eosinophils undergo ETosis and loss of galectin-10 cytoplasmic localization results in intracellular CLC formation. Free galectin-10 released following plasma membrane disintegration forms extracellular CLCs. Of interest, galectin-10-containing extracellular vesicles are also released during ETosis. Mice models indicated that CLCs could be a novel therapeutic target for Th2-type airway inflammation. The concept of ETosis, which represents a major fate of activated eosinophils, expands our current understanding by which cytoplasmic galectin-10 is crystalized/externalized. Besides CLCs and free galectin-10, cell-free granules, extracellular chromatin traps, extracellular vesicles, and other alarmins, all released through the process of ETosis, have novel implications in various eosinophilic disorders.
Topics: Animals; Crystallization; Disease Models, Animal; Eosinophilia; Extracellular Traps; Galectins; Humans; Inflammation; Mice
PubMed: 31203469
DOI: 10.1007/s11882-019-0868-0 -
Toxins Jun 2021The development of finely tuned and reliable crystallization processes to obtain crystalline formulations of proteins has received growing interest from different... (Review)
Review
The development of finely tuned and reliable crystallization processes to obtain crystalline formulations of proteins has received growing interest from different scientific fields, including toxinology and structural biology, as well as from industry, notably for biotechnological and medical applications. As a natural crystal-making bacterium, () has evolved through millions of years to produce hundreds of highly structurally diverse pesticidal proteins as micrometer-sized crystals. The long-term stability of protein crystals in aqueous environments and their specific and controlled dissolution are characteristics that are particularly sought after. In this article, I explore whether the crystallization machinery of can be hijacked as a means to produce (micro)crystalline formulations of proteins for three different applications: (i) to develop new bioinsecticidal formulations based on rationally improved crystalline toxins, (ii) to functionalize crystals with specific characteristics for biotechnological and medical applications, and (iii) to produce microcrystals of custom proteins for structural biology. By developing the needs of these different fields to figure out if and how could meet each specific requirement, I discuss the already published and/or patented attempts and provide guidelines for future investigations in some underexplored yet promising domains.
Topics: Bacillus thuringiensis; Bacillus thuringiensis Toxins; Bacterial Proteins; Crystallization; Pest Control, Biological
PubMed: 34206749
DOI: 10.3390/toxins13070441 -
International Journal of Molecular... Aug 2022This review is aimed to provide to an "educated but non-expert" readership and an overview of the scientific, commercial, and ethical importance of investigating the... (Review)
Review
This review is aimed to provide to an "educated but non-expert" readership and an overview of the scientific, commercial, and ethical importance of investigating the crystalline forms (polymorphs, hydrates, and co-crystals) of active pharmaceutical ingredients (API). The existence of multiple crystal forms of an API is relevant not only for the selection of the best solid material to carry through the various stages of drug development, including the choice of dosage and of excipients suitable for drug development and marketing, but also in terms of intellectual property protection and/or extension. This is because the physico-chemical properties, such as solubility, dissolution rate, thermal stability, processability, etc., of the solid API may depend, sometimes dramatically, on the crystal form, with important implications on the drug's ultimate efficacy. This review will recount how the scientific community and the pharmaceutical industry learned from the catastrophic consequences of the appearance of new, more stable, and unsuspected crystal forms. The relevant aspects of hydrates, the most common pharmaceutical solid solvates, and of co-crystals, the association of two or more solid components in the same crystalline materials, will also be discussed. Examples will be provided of how to tackle multiple crystal forms with screening protocols and theoretical approaches, and ultimately how to turn into discovery and innovation the purposed preparation of new crystalline forms of an API.
Topics: Crystallization; Excipients; Pharmaceutical Preparations; Solubility
PubMed: 36012275
DOI: 10.3390/ijms23169013