Authors: Moshe A Dessau, Yorgo Modis

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

Authors: Simon Weiss, Sandra Vergara, Guowu Lin...

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

Review

Authors: Isabelle Martiel, Henrike M Müller-Werkmeister, Aina E Cohen...

Highly efficient data-collection methods are required for successful macromolecular crystallography (MX) experiments at X-ray free-electron lasers (XFELs). XFEL beamtime is scarce, and the high peak brightness of each XFEL pulse destroys the exposed crystal volume. It is therefore necessary to combine diffraction images from a large number of crystals (hundreds to hundreds of thousands) to obtain a final data set, bringing about sample-refreshment challenges that have previously been unknown to the MX synchrotron community. In view of this experimental complexity, a number of sample delivery methods have emerged, each with specific requirements, drawbacks and advantages. To provide useful selection criteria for future experiments, this review summarizes the currently available sample delivery methods, emphasising the basic principles and the specific sample requirements. Two main approaches to sample delivery are first covered: (i) injector methods with liquid or viscous media and (ii) fixed-target methods using large crystals or using microcrystals inside multi-crystal holders or chips. Additionally, hybrid methods such as acoustic droplet ejection and crystal extraction are covered, which combine the advantages of both fixed-target and injector approaches.

Topics: Acoustics; Animals; Crystallization; Crystallography, X-Ray; Electrons; Equipment Design; Flow Injection Analysis; Humans; Lasers; Proteins; Time Factors

PubMed: 30821705
DOI: 10.1107/S2059798318017953

Review

Authors: John R G Sander, Brad W Zeiger, Kenneth S Suslick...

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

Authors: Fabian von Rohr, Andreas Schilling, Robert J Cava...

The thermoelectric properties between 10 and 300 K and the growth of single crystals of n-type and p-type GeBi(4)Te(7), GeSb(4)Te(7) and Ge(Bi(1-x)Sb(x))(4)Te(7) solid solution are reported. Single crystals were grown by the modified Bridgman method, and p-type behavior was achieved by the substitution of Bi by Sb in GeBi(4)Te(7). The thermopower in the Ge(Bi(1-x)Sb(x))(4)Te(7) solid solution ranges from -117 to +160 μV K(-1). The crossover from n-type to p-type is continuous with increasing Sb content and is observed at x ≈0.15. The highest thermoelectric efficiencies among the tested n-type and p-type samples are Z(n)T = 0.11 and Z(p)T = 0.20, respectively. For an optimal n-p couple in this alloy system the composite figure of merit is Z(np)T = 0.17 at room temperature.

Topics: Crystallization; Electric Conductivity; Energy Transfer; Germanium; Materials Testing; Semiconductors; Temperature

PubMed: 23343638
DOI: 10.1088/0953-8984/25/7/075804

Review

Authors: Nasrul Wathoni, Wuri Ariestika Sari, Khaled M Elamin...

Most recently discovered active pharmaceutical molecules and market-approved medicines are poorly soluble in water, resulting in limited drug bioavailability and therapeutic effectiveness. The application of coformers in a multicomponent crystal method is one possible strategy to modulate a drug's solubility. A multicomponent crystal is a solid phase formed when several molecules of different substances crystallize in a crystal lattice with a certain stoichiometric ratio. The goal of this review paper is to comprehensively describe the application of coformers in the formation of multicomponent crystals as solutions for pharmaceutically active ingredients with limited solubility. Owing to their benefits including improved physicochemical profile of pharmaceutically active ingredients, multicomponent crystal methods are predicted to become increasingly prevalent in the development of active drug ingredients in the future.

Topics: Crystallization; Solubility; Biological Availability; Water; Pharmaceutical Preparations

PubMed: 36557827
DOI: 10.3390/molecules27248693

Review

Authors: Dario Braga, Lucia Casali, Fabrizia Grepioni...

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

Review

Authors: Robin L Owen, Jordi Juanhuix, Martin Fuchs...

Following pioneering work 40 years ago, synchrotron beamlines dedicated to macromolecular crystallography (MX) have improved in almost every aspect as instrumentation has evolved. Beam sizes and crystal dimensions are now on the single micron scale while data can be collected from proteins with molecular weights over 10 MDa and from crystals with unit cell dimensions over 1000 Å. Furthermore it is possible to collect a complete data set in seconds, and obtain the resulting structure in minutes. The impact of MX synchrotron beamlines and their evolution is reflected in their scientific output, and MX is now the method of choice for a variety of aims from ligand binding to structure determination of membrane proteins, viruses and ribosomes, resulting in a much deeper understanding of the machinery of life. A main driving force of beamline evolution have been advances in almost every aspect of the instrumentation comprising a synchrotron beamline. In this review we aim to provide an overview of the current status of instrumentation at modern MX experiments. The most critical optical components are discussed, as are aspects of endstation design, sample delivery, visualisation and positioning, the sample environment, beam shaping, detectors and data acquisition and processing.

Topics: Crystallization; Crystallography; Equipment Design; Equipment Failure Analysis; Multiprotein Complexes; Synchrotrons

PubMed: 27046341
DOI: 10.1016/j.abb.2016.03.021

Review

Authors: Alexander McPherson, Bob Cudney

For the successful X-ray structure determination of macromolecules, it is first necessary to identify, usually by matrix screening, conditions that yield some sort of crystals. Initial crystals are frequently microcrystals or clusters, and often have unfavorable morphologies or yield poor diffraction intensities. It is therefore generally necessary to improve upon these initial conditions in order to obtain better crystals of sufficient quality for X-ray data collection. Even when the initial samples are suitable, often marginally, refinement of conditions is recommended in order to obtain the highest quality crystals that can be grown. The quality of an X-ray structure determination is directly correlated with the size and the perfection of the crystalline samples; thus, refinement of conditions should always be a primary component of crystal growth. The improvement process is referred to as optimization, and it entails sequential, incremental changes in the chemical parameters that influence crystallization, such as pH, ionic strength and precipitant concentration, as well as physical parameters such as temperature, sample volume and overall methodology. It also includes the application of some unique procedures and approaches, and the addition of novel components such as detergents, ligands or other small molecules that may enhance nucleation or crystal development. Here, an attempt is made to provide guidance on how optimization might best be applied to crystal-growth problems, and what parameters and factors might most profitably be explored to accelerate and achieve success.

Topics: Animals; Crystallization; Crystallography, X-Ray; Humans; Macromolecular Substances; Osmolar Concentration

PubMed: 25372810
DOI: 10.1107/S2053230X14019670

Review

Authors: Zbigniew Dauter

Diffraction data acquisition is the final experimental stage of the crystal structure analysis. All subsequent steps involve mainly computer calculations. Optimally measured and accurate data make the structure solution and refinement easier and lead to more faithful interpretation of the final models. Here, the important factors in data collection from macromolecular crystals are discussed and strategies appropriate for various applications, such as molecular replacement, anomalous phasing, and atomic-resolution refinement are presented. Criteria useful for judging the diffraction data quality are also discussed.

Topics: Crystallization; Crystallography, X-Ray; Data Interpretation, Statistical; Image Processing, Computer-Assisted; Protein Conformation; Proteins; Synchrotrons; X-Ray Diffraction

PubMed: 28573573
DOI: 10.1007/978-1-4939-7000-1_7