A crystal-processing machine using a deep-ultraviolet laser: application to long-wavelength native SAD experiments.

While native SAD phasing is a promising method for next-generation macromolecular crystallography, it requires the collection of high-quality diffraction data using long-wavelength X-rays. The crystal itself and the noncrystalline medium around the crystal can cause background noise during long-wavelength X-ray data collection, hampering native SAD phasing. Optimizing the crystal size and shape or removing noncrystalline sample portions have thus been considered to be effective means of improving the data quality. A crystal-processing machine that uses a deep-UV laser has been developed. The machine utilizes the pulsed UV laser soft ablation (PULSA) technique, which generates less heat than methods using infrared or visible lasers. Since protein crystals are sensitive to heat damage, PULSA is an appropriate method to process them. Integration of a high-speed Galvano scanner and a high-precision goniometer enables protein crystals to be shaped precisely and efficiently. Application of this crystal-processing machine to a long-wavelength X-ray diffraction experiment significantly improved the diffraction data quality and thereby increased the success rate in experimental phasing using anomalous diffraction from atoms.

Pharmaceutical co-crystals of posaconazole for improvement of physicochemical properties

Abstract Posaconazole exerts an extended spectrum of antifungal activity against various strains of clinically relevant moulds and yeasts. In recent years, antifungal triazole posaconazole has become increasingly important for the prophylaxis and treatment of systemic mycoses. After oral administration of posaconazole, absolute bioavailability has been estimated to range from 8% to 47%. Pharmaceutical co-crystallization is a promising approach for improving dissolution rate or manipulating other physical properties of API. The objective of this study is to improve the dissolution rate of posaconazole by co-crystallization. A 1:1 stoichiometric co-crystals of adipic acid were prepared by solvent assisted grinding method. The prepared co-crystals were subjected to solid-state characterization by FTIR, PXRD and DSC studies. The physicochemical properties of posaconazole and co-crystals were assessed in terms of melting point, flowability and dissolution rate. The results indicated improvement in flow property and dissolution rate. In vitro dissolution profile of co-crystals showed a significant increased dissolution of posaconazole from initial period in 0.1 N hydrochloric acid solution. The dissolution efficiency for posaconazole-adipic acid co-crystal was 61.65 % against posaconazole, 46.58 %. Thus, co-crystallization can be a promising approach to prepare posaconazole-adipic acid co-crystals with improved physicochemical properties.

Efavirenz dissolution enhancement V - A combined top down/bottom up approach on nanocrystals formulation

Abstract Efavirenz is one of the most commonly used drugs in HIV therapy. However the low water solubility tends to result in low bioavailability. Drug nanocrystals, should enhance the dissolution and consequently bioavailability. The aim of the present study was to obtain EFV nanocrystals prepared by an antisolvent technique and to further observe possible effect, on the resulting material, due to altering crystallization parameters. A solution containing EFV and a suitable solvent was added to an aqueous solution of particle stabilizers, under high shear agitation. Experimental conditions such as solvent/antisolvent ratio; drug load; solvent supersaturation; change of stabilizer; addition of milling step and solvents of different polarities were evaluated. Suspensions were characterized by particle size and zeta potential. After freeze- dried and the resulting powder was characterized by PXRD, infrared spectroscopy and SEM. Also dissolution profiles were obtained. Many alterations were not effective for enhancing EFV dissolution; some changes did not even produced nanosuspensions while other generated a different solid phase from the polymorph of raw material. Nevertheless reducing EFV load produced enhancement on dissolution profile. The most important modification was adding a milling step after precipitation. The resulting suspension was more uniform and the powder presented grater enhancement of dissolution efficacy.

Novel combined crystallization plate for high-throughput crystal screening and in situ data collection at a crystallography beamline.

In situ microplates are small in size, crystal cultivation and operation are difficult, and the efficiency of crystal screening is relatively low. To solve this problem, a novel combined crystallization plate was designed for high-throughput crystal cultivation and in situ data collection. A frame was used to hold 48 in situ microplates, and the in situ microplates were sealed on one side with an ultralow background-scattering Kapton film. An automatic liquid handler (Mosquito) was used to add a liquid drop to the in situ microplates in the frame, and CrystalClear HD tape was used to seal the frame. A sealed frame holding 48 microplates was developed as a novel combined crystallization plate and was used for crystal cultivation under different conditions and in situ data collection at the synchrotron beamline. Moreover, individual microplates can be separated from the combined crystal plate and then fixed on a magnetic base or loaded onto a UniPuck for in situ data collection. Automatic grid scanning was used to locate crystals. The efficiency of the combined crystallization plate for crystal screening was verified. This method avoids the manual manipulation of crystals during crystal screening and diffraction data collection; therefore, the combined crystallization plate is suitable for large-scale screening of microcrystals.

A fully automated crystallization apparatus for small protein quantities.

In 2003, a fully automated protein crystallization and monitoring system (PXS) was developed to support the structural genomics projects that were initiated in the early 2000s. In PXS, crystallization plates were automatically set up using the vapor-diffusion method, transferred to incubators and automatically observed according to a pre-set schedule. The captured images of each crystallization drop could be monitored through the internet using a web browser. While the screening throughput of PXS was very high, the demands of users have gradually changed over the ensuing years. To study difficult proteins, it has become important to screen crystallization conditions using small amounts of proteins. Moreover, membrane proteins have become one of the main targets for X-ray crystallography. Therefore, to meet the evolving demands of users, PXS was upgraded to PXS2. In PXS2, the minimum volume of the dispenser is reduced to 0.1 µl to minimize the amount of sample, and the resolution of the captured images is increased to five million pixels in order to observe small crystallization drops in detail. In addition to the 20°C incubators, a 4°C incubator was installed in PXS2 because crystallization results may vary with temperature. To support membrane-protein crystallization, PXS2 includes a procedure for the bicelle method. In addition, the system supports a lipidic cubic phase (LCP) method that uses a film sandwich plate and that was specifically designed for PXS2. These improvements expand the applicability of PXS2, reducing the bottleneck of X-ray protein crystallography.

Polysaccharide-Based Injection Matrix for Serial Crystallography.

Serial crystallography (SX) provides an opportunity to observe the molecular dynamics of macromolecular structures at room temperature via pump-probe studies. The delivery of crystals embedded in a viscous medium via an injector or syringe is widely performed in synchrotrons or X-ray free-electron laser facilities with low repetition rates. Various viscous media have been developed; however, there are cases in which the delivery material undesirably interacts chemically or biologically with specific protein samples, or changes the stability of the injection stream, depending on the crystallization solution. Therefore, continued discovery and characterization of new delivery media is necessary for expanding future SX applications. Here, the preparation and characterization of new polysaccharide (wheat starch (WS) and alginate)-based sample delivery media are introduced for SX. Crystals embedded in a WS or alginate injection medium showed a stable injection stream at a flow rate of < 200 nL/min and low-level X-ray background scattering similar to other hydrogels. Using these media, serial millisecond crystallography (SMX) was performed, and the room temperature crystal structures of glucose isomerase and lysozyme were determined at 1.9-2.0 Å resolutions. WS and alginate will allow an expanded application of sample delivery media in SX experiments.

Remote Controlled Autonomous Microgravity Lab Platforms for Drug Research in Space.

Research conducted in microgravity conditions has the potential to yield new therapeutics, as advances can be achieved in the absence of phenomena such as sedimentation, hydrostatic pressure and thermally-induced convection. The outcomes of such studies can significantly contribute to many scientific and technological fields, including drug discovery. This article reviews the existing traditional microgravity platforms as well as emerging ideas for enabling microgravity research focusing on SpacePharma's innovative autonomous remote-controlled microgravity labs that can be launched to space aboard nanosatellites to perform drug research in orbit. The scientific literature is reviewed and examples of life science fields that have benefited from studies in microgravity conditions are given. These include the use of microgravity environment for chemical applications (protein crystallization, drug polymorphism, self-assembly of biomolecules), pharmaceutical studies (microencapsulation, drug delivery systems, behavior and stability of colloidal formulations, antibiotic drug resistance), and biological research, including accelerated models for aging, investigation of bacterial virulence , tissue engineering using organ-on-chips in space, enhanced stem cells proliferation and differentiation.

Optical Processing of DNA-Programmed Nanoparticle Superlattices.

Hierarchical structural control across multiple size regimes requires careful consideration of the complex energy- and time-scales which govern the system's morphology at each of these different size ranges. At the nanoscale, synthetic chemistry techniques have been developed to create nanoparticles of well-controlled size and composition. At the macroscale, it is feasible to directly impose material structure via physical manipulation. However, in between these two size regimes at the mesoscale, structural control is more challenging as the physical forces that govern material assembly at larger and smaller scales begin to interfere with one another. In this work, the interplay of structure-directing forces at multiple length-scales is investigated by utilizing optical processing to influence both nanoscale and microscale features of self-assembled, DNA-grafted nanoparticle films. Optical processing is used to generate heat, which causes the self-assembled particles to rearrange from a kinetically trapped, amorphous state into a thermodynamically preferred superlattice structure. The gradient in the heat profile, however, also induces thermophoretic motion within the nanoparticle film, resulting in microscale movement at a comparable time-scale. By utilizing precise exposure times enabled by optical processing, crystallization and thermophoresis occur concurrently in the self-assembling nanoparticle system, enabling a dynamic growth mechanism whereby nucleation and growth occur in separate regions of the material. Furthermore, utilizing sufficiently short processing times allows for the formation of a fluidlike state of the DNA-functionalized nanoparticle materials that is inaccessible via typical thermal processing setups. This unique phase of the material allows for both pathway-dependent and pathway-independent growth phenomena, as appropriately tuning the experimental conditions enables the formation of morphologically equivalent nanoparticle lattices that are generated through different intermediate states (pathway-independent structures), or kinetically preprocessing a material to yield unique thermodynamic arrangements of particles once fully annealed (pathway-dependent structures).

Enhancing pharmaceutical crystallization in a flow crystallizer with ultrasound: Anti-solvent crystallization.

Continuous crystallization is a fast growing application domain in the pharmaceutical industry. Application of ultrasound has been proven to have positive effects like reduction in induction time and Metastable Zone Width (MSZW) in both batch and flow systems. Further understanding of flow-based sonocrystallization is required to achieve industrial level scale up. This work investigates the sonocrystallization of pharmaceutical compounds in a tubular flow crystallizer. Acetyl Salicylic Acid (ASA-Aspirin) is used as a model compound with ethanol and water as solvent and anti-solvent, respectively. Experiments were conducted in silent and sonicated conditions to study the MSZW. Ultrasound made it possible to achieve crystallization within the crystallizer which was not possible in silent conditions, under the tested conditions. Continuous crystallization was achieved at as low as 48 wt% of anti-solvent and crystallization was already seen at a supersaturation of 1.02. In some experiments, temperature rise with ultrasound caused the crystals to re-dissolve within the channels. Better crystallization - no re-dissolution - was achieved by using low ultrasonic power without any loss in the yield. Particle sizes of product crystals were in the range of 4-46 µm. In conclusion, ultrasound was highly effective in enabling anti-solvent crystallization of a pharmaceutical compound in a tubular flow crystallizer.

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering.

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. A prerequisite for the method is that highly ordered crystalline specimen need to be grown from the macromolecule to be studied, which then need to be prepared for the diffraction experiment. This preparation procedure typically involves removal of the crystal from the solution, in which it was grown, soaking of the crystal in ligand solution or cryo-protectant solution and then immobilizing the crystal on a mount suitable for the experiment. A serious problem for this procedure is that macromolecular crystals are often mechanically unstable and rather fragile. Consequently, the handling of such fragile crystals can easily become a bottleneck in a structure determination attempt. Any mechanical force applied to such delicate crystals may disturb the regular packing of the molecules and may lead to a loss of diffraction power of the crystals. Here, we present a novel all-in-one sample holder, which has been developed in order to minimize the handling steps of crystals and hence to maximize the success rate of the structure determination experiment. The sample holder supports the setup of crystal drops by replacing the commonly used microscope cover slips. Further, it allows in-place crystal manipulation such as ligand soaking, cryo-protection and complex formation without any opening of the crystallization cavity and without crystal handling. Finally, the sample holder has been designed in order to enable the collection of in situ X-ray diffraction data at both, ambient and cryogenic temperature. By using this sample holder, the chances to damage the crystal on its way from crystallization to diffraction data collection are considerably reduced since direct crystal handling is no longer required.

Charcot-Leyden Crystals in Eosinophilic Inflammation: Active Cytolysis Leads to Crystal Formation.

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.

Microfluidic sample delivery for serial crystallography using XFELs.

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) is an emerging field for structural biology. One of its major impacts lies in the ability to reveal the structure of complex proteins previously inaccessible with synchrotron-based crystallography techniques and allowing time-resolved studies from femtoseconds to seconds. The nature of this serial technique requires new approaches for crystallization, data analysis, and sample delivery. With continued advancements in microfabrication techniques, various developments have been reported in the past decade for innovative and efficient microfluidic sample delivery for crystallography experiments using XFELs. This article summarizes the recent developments in microfluidic sample delivery with liquid injection and fixed-target approaches, which allow exciting new research with XFELs. Graphical abstract.

Resolving polymorphs and radiation-driven effects in microcrystals using fixed-target serial synchrotron crystallography.

The ability to determine high-quality, artefact-free structures is a challenge in micro-crystallography, and the rapid onset of radiation damage and requirement for a high-brilliance X-ray beam mean that a multi-crystal approach is essential. However, the combination of crystal-to-crystal variation and X-ray-induced changes can make the formation of a final complete data set challenging; this is particularly true in the case of metalloproteins, where X-ray-induced changes occur rapidly and at the active site. An approach is described that allows the resolution, separation and structure determination of crystal polymorphs, and the tracking of radiation damage in microcrystals. Within the microcrystal population of copper nitrite reductase, two polymorphs with different unit-cell sizes were successfully separated to determine two independent structures, and an X-ray-driven change between these polymorphs was followed. This was achieved through the determination of multiple serial structures from microcrystals using a high-throughput high-speed fixed-target approach coupled with robust data processing.

Long-wavelength Mesh&Collect native SAD phasing from microcrystals.

Harnessing the anomalous signal from macromolecular crystals with volumes of less than 10 000 µm3 for native phasing requires careful experimental planning. The type of anomalous scatterers that are naturally present in the sample, such as sulfur, phosphorus and calcium, will dictate the beam energy required and determine the level of radiation sensitivity, while the crystal size will dictate the beam size and the sample-mounting technique, in turn indicating the specifications of a suitable beamline. On the EMBL beamline P13 at PETRA III, Mesh&Collect data collection from concanavalin A microcrystals with linear dimensions of ∼20 µm or less using an accordingly sized microbeam at a wavelength of 1.892 Š(6.551 keV, close to the Mn edge at 6.549 keV) increases the expected Bijvoet ratio to 2.1% from an expected 0.7% at 12.6 keV (Se K edge), thus allowing experimental phase determination using the anomalous signal from naturally present Mn2+ and Ca2+ ions. Dozens of crystals were harvested and flash-cryocooled in micro-meshes, rapidly screened for diffraction (less than a minute per loop) and then used for serial Mesh&Collect collection of about 298 partial data sets (10° of crystal rotation per sample). The partial data sets were integrated and scaled. A genetic algorithm for combining partial data sets was used to select those to be merged into a single data set. This final data set showed high completeness, high multiplicity and sufficient anomalous signal to locate the anomalous scatterers, and provided phasing information which allowed complete auto-tracing of the polypeptide chain. To allow the complete experiment to run in less than 2 h, a practically acceptable time frame, the diffractometer and detector had to run together with limited manual intervention. The combination of several cutting-edge components allowed accurate anomalous signal to be measured from small crystals.

Strategies for sample delivery for femtosecond crystallography.

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.

X-Ray Free-Electron Lasers for the Structure and Dynamics of Macromolecules.

X-ray free-electron lasers provide femtosecond-duration pulses of hard X-rays with a peak brightness approximately one billion times greater than is available at synchrotron radiation facilities. One motivation for the development of such X-ray sources was the proposal to obtain structures of macromolecules, macromolecular complexes, and virus particles, without the need for crystallization, through diffraction measurements of single noncrystalline objects. Initial explorations of this idea and of outrunning radiation damage with femtosecond pulses led to the development of serial crystallography and the ability to obtain high-resolution structures of small crystals without the need for cryogenic cooling. This technique allows the understanding of conformational dynamics and enzymatics and the resolution of intermediate states in reactions over timescales of 100 fs to minutes. The promise of more photons per atom recorded in a diffraction pattern than electrons per atom contributing to an electron micrograph may enable diffraction measurements of single molecules, although challenges remain.

Application of In Situ Product Crystallization and Related Techniques in Biocatalytic Processes.

This Minireview highlights the application of crystallization as a very powerful in situ product removal (ISPR) technique in biocatalytic process design. Special emphasis is placed on its use for in situ product crystallization (ISPC) to overcome unfavorable thermodynamic reaction equilibria, inhibition, and undesired reactions. The combination of these unit operations requires an interdisciplinary perspective to find a holistic solution for the underlying bioprocess intensification approach. Representative examples of successful integrated process options are selected, presented, and assessed regarding their overall productivity and applicability. In addition, parallels to the use of adsorption as a very similar technique are drawn and similarities discussed.

Using sound pulses to solve the crystal-harvesting bottleneck.

Crystal harvesting has proven to be difficult to automate and remains the rate-limiting step for many structure-determination and high-throughput screening projects. This has resulted in crystals being prepared more rapidly than they can be harvested for X-ray data collection. Fourth-generation synchrotrons will support extraordinarily rapid rates of data acquisition, putting further pressure on the crystal-harvesting bottleneck. Here, a simple solution is reported in which crystals can be acoustically harvested from slightly modified MiTeGen In Situ-1 crystallization plates. This technique uses an acoustic pulse to eject each crystal out of its crystallization well, through a short air column and onto a micro-mesh (improving on previous work, which required separately grown crystals to be transferred before harvesting). Crystals can be individually harvested or can be serially combined with a chemical library such as a fragment library.

A high-throughput multi-microfluidic crystal generator (MMicroCryGen) platform for facile screening of polymorphism and crystal morphology for pharmaceutical compounds.

In this work, a novel multi-microfluidic crystallization platform called MMicroCryGen is presented, offering a facile methodology for generating individual crystals for fast and easy screening of the polymorphism and crystal habit of solid compounds. The MMicroCryGen device is capable of performing 8 × 10 cooling crystallization experiments in parallel using 8 disposable microcapillary film strips, each requiring less than 25 µL of solution. Compared to traditional microfluidic systems, the MMicroCryGen platform does not require complex fluid handling; it can be directly integrated with a 96-well microplate and it can also work in a "dipstick" mode. The produced crystals can be safely and directly observed inside the capillaries by optical and spectroscopic techniques. The platform was validated by performing a number of independent experimental runs for: (1) polymorph and hydrate screening of ortho-aminobenzoic acid, succinic acid and piroxicam; (2) co-crystal form screening of the p-toluenesulfonamide/triphenylphosphine oxide system; (3) studying the effect of o-toluic acid on ortho-aminobenzoic cooling crystallization (effect of structurally related additives). In all three cases, all known solid forms were identified with a single experiment using ∼200 µL of solvent and just a few micrograms of the solid material. The MMicroCryGen is simple to use, inexpensive and it provides increased flexibility compared to traditional crystallization techniques, being an effective new microfluidic solution for solid form screening in pharmaceutical, fine chemicals, food and agrochemical industries.

X-Ray Crystallographic Studies for Revealing Binding Sites of General Anesthetics in Pentameric Ligand-Gated Ion Channels.

X-ray crystallography is a powerful tool in structural biology and can offer insight into structured-based understanding of general anesthetic action on various relevant molecular targets, including pentameric ligand-gated ion channels (pLGICs). In this chapter, we outline the procedures for expression and purification of pLGICs. Optimization of crystallization conditions, especially to achieve high-resolution structures of pLGICs bound with general anesthetics, is also presented. Case studies of pLGICs bound with the volatile general anesthetic isoflurane, 2-bromoethanol, and the intravenous general anesthetic ketamine are revisited.