ChapterPDF AvailableLiterature ReviewAdvanced Methods of Protein CrystallizationJune 2017Methods in Molecular Biology 1607:51-76DOI:10.1007/978-1-4939-7000-1_3In book: Protein Crystallography (pp.51-76)Project: CrystallogenesisAuthors: Abel MorenoUniversidad Nacional Autónoma de México Download full-text PDFRead full-textDownload full-text PDFRead full-textDownload citation Copy link Link copied Read full-text Download citation Copy link Link copiedCitations (8)References (190)Figures (9)Abstract and FiguresThis chapter provides a review of different advanced methods that help to increase the success rate of a crystallization project, by producing larger and higher quality single crystals for determination of macromolecular structures by crystallographic methods. For this purpose, the chapter is divided into three parts. The first part deals with the fundamentals for understanding the crystallization process through different strategies based on physical and chemical approaches. The second part presents new approaches involved in more sophisticated methods not only for growing protein crystals but also for controlling the size and orientation of crystals through utilization of electromagnetic fields and other advanced techniques. The last section deals with three different aspects: the importance of microgravity, the use of ligands to stabilize proteins, and the use of microfluidics to obtain protein crystals. All these advanced methods will allow the readers to obtain suitable crystalline samples for high-resolution X-ray and neutron crystallography. A scheme representing different methods used to crystallize proteins. The classical methods are shown on the left, and the advanced methods, usually called nonconventional methods of protein crystallization, are shown on the right…  a) The solubility phase diagram (also known as Oswald-Miers diagram) is divided into different zones: undersaturated, supersaturated, metastable, nucleation, and precipitation. (b) The energetics of the system is very important to understand; it expresses the kinetics of the crystallization and allows to predict the critical size of the nucleus to be converted into crystal…  Different designs of e-crystallization growth-cell for applying electric field to the crystallization process of biological macromolecules. The two frames on the left are useful for batch crystallization setup, and the one on the right is for a vapor diffusion setup…  Two pieces of apparatus used for e-crystallization of proteins: (a) for applying DC ranging from 2-6 μA, (b) for applying AC during the crystallization of proteins. First, nucleation is induced and then the crystal growth proceeds via vapor diffusion…  +4Crystals of glucose isomerase grown: (a) when a direct current (DC) of 2 ?A is applied for 48 h and subsequently the crystal growth proceeds in 2 weeks by the sitting-drop setup, and (b) when an alternant current (AC) of 2 and 8 Hz is applied for 48 h. The bar scale for (b) is the same at 2 and 8 Hz… Figures - uploaded by Abel MorenoAuthor contentAll figure content in this area was uploaded by Abel MorenoContent may be subject to copyright. Discover the world s research20+ million members135+ million publications700k+ research projectsJoin for freePublic Full-text 1Content uploaded by Abel MorenoAuthor contentAll content in this area was uploaded by Abel Moreno on Nov 11, 2017 Content may be subject to copyright. 51Alexander Wlodawer et al. (eds.), Protein Crystallography: Methods and Protocols, Methods in Molecular Biology, vol. 1607,DOI 10.1007/978-1-4939-7000-1_3, © Springer Science+Business Media LLC 2017Chapter 3Advanced Methods of Protein CrystallizationAbel MorenoAbstractThis chapter provides a review of different advanced methods that help to increase the success rate of a crystallization project, by producing larger and higher quality single crystals for determination of macro-molecular structures by crystallographic methods. For this purpose, the chapter is divided into three parts. The first part deals with the fundamentals for understanding the crystallization process through different strategies based on physical and chemical approaches. The second part presents new approaches involved in more sophisticated methods not only for growing protein cr ystals but also for controlling the size and orientation of crystals through utilization of electromagnetic fields and other advanced techniques. The last section deals with three different aspects: the importance of microgravity, the use of ligands to stabilize proteins, and the use of microfluidics to obtain protein crystals. All these advanced methods will allow the readers to obtain suitable crystalline samples for high-resolution X-ray and neutron crystallography.Key words Electric fields, Magnetic fields, Counter-diffusion techniques, Crystal growth in gels, Protein crystallization1 IntroductionProteins, nucleic acids, polysaccharides, and lipids are regarded as the most important molecules of life. The function of these mole-cules in sustaining life depends on their three-dimensional struc-ture and on their highly specific mutual interactions, dictated by their structure and bonding properties [1]. Getting to know the structures of macromolecules and of their complexes will enhance our understanding of biological processes of life. It will also hint at novel ways to treat a wide range of diseases, from congenital anom-alies through bacterial and viral infections to autoimmunity dis-eases [2], or even many different types of cancers [3, 4]. X-ray crystallography is the hallmark of this search (it is the most power-ful technique for structure elucidation of macromolecules), as it reaches near-atomic resolution in the most favorable cases, without a priori limitation on the size or on the complexity of the studied molecules. X-ray crystallography requires the growth of large and well-diffracting crystals (for conventional crystallography) or 52nanocrystals (for free electron lasers, XFELs). The production of such crystals is the most intractable stage in the process of struc-ture determination [5, 6].There are a number of strategies, from classical techniques to advanced methods, that focus on obtaining high quality single crys-tals (Fig. 1) for high resolution crystallographic analyses. Despite the existence of a large variety of conventional crystallization tech-niques (see Chapters 2 and 4 by McPherson and Derewenda) and the automation of high-throughput screening systems, statistics from various structural programs indicate that only fewer than 20% of de novo overexpressed proteins yield diffracting crystals [7]. This represents a very low success rate considering the cumulative diffi-culties of cloning, expressing, and purifying proteins. Although we cannot fully identify why some proteins do not crystallize, this may be due to the intrinsic physico- chemical properties of the protein per se. For this reason, it will be useful to have user-friendly tools that allow the experimenter to a priori select successful protein tar-gets for crystallization and for identifying problematic proteins. The proteins that are recalcitrant to crystallization can be highly flexible as well as completely unstructured. They will not nucleate properly for different reasons, such as propensity to aggregate in an amorphous phase or difficulty to form stable crystal contacts. Therefore, obtaining good crystals can be very tricky and often needs a combination of strategies such as protein engineering, sophisticated crystallization techniques, and a good understanding of the nucleation and crystal growth processes [8–10].Fig. 1 A scheme representing different methods used to crystallize proteins. The classical methods are shown on the left, and the advanced methods, usually called nonconventional methods of protein crystallization, are shown on the rightAbel Moreno 53In this chapter different advanced methods that help to increase the success rate of a crystallization project in order to obtain high quality single crystals for crystallographic research are discussed. The first part presents the body of knowledge regarding the crys-tallization process from physical and chemical perspectives. The second part introduces the reader to new approaches related to more sophisticated methods, not only for growing protein crystals but also for controlling the crystal size and orientation by electro-magnetic fields, as well as through other advanced methods. Additional information including the importance of microgravity, the use of ligands to stabilize proteins, and the use of microfluidics to obtain suitable protein crystals for high-resolution X-ray crystal-lography, is also presented.2 Technical ApproachesThe solubility diagrams and the energetics of nucleation (Fig. 2a, b, respectively) provide vital and necessary information for the optimization of crystal growth [11–13]. In most cases, their use will lead to a reasonable strategy for obtaining protein crystals and for assuring high reproducibility. Crystal nucleation occurs in two stages: nucleation of new crystal embryos, and growth of a few nuclei into full-size diffracting crystals (Fig. 2b). It has been shown that the optimal conditions for growing high-quality crys-tals (large size, and minimum of imperfections) involve lower macromolecule supersaturation levels than those required for ini-tial nucleation [14, 15]. Nucleation cannot take place at these lower supersaturations because an energy barrier of kinetic origin 2.1 Fundamentals of Protein Crystallization Process Applied to Advanced Methods of Protein Crystal GrowthFig. 2 (a) The solubility phase diagram (also known as Oswald-Miers diagram) is divided into different zones: undersaturated, supersaturated, metastable, nucleation, and precipitation. (b) The energetics of the system is very important to understand; it expresses the kinetics of the crystallization and allows to predict the critical size of the nucleus to be converted into crystalAdvanced Protein Crystallization 54(due to the energetically expensive formation of the crystal–solu-tion interface) is involved [16, 17]. The establishment of crystal-lization solubility phase diagrams allows precise identification of the limits between the spontaneous nucleation and the optimal growth (often called \"metastable”) zones [18, 19]. That informa-tion can subsequently be used for growing crystals as close as pos-sible to the metastable zone or for incubating the trials at nucleation conditions for a time sufficient for the formation of a few nuclei before transiting to metastable conditions for optimal growth (by changing the concentration of the precipitating agent, pH, or temperature) [18, 20–22].There are alternative setup techniques such as microbatch under oil [23] or crystallization in capillaries [24, 25]. Often, these alternatives produce crystals under screening conditions that will be difficult to produce with other setups (e.g., standard vapor dif-fusion). These alternative techniques can also produce higher- quality crystals. Each technique relies on a different geometry and different way to reach supersaturation, therefore they present a kinetically different situation. These subtle differences frequently lead to different results in an unpredictable way. Tiny crystals of the same protein can start the nucleation process. There are various crystal seeding techniques, including the standard microseeding and streak-seeding into metastable conditions using microcrystals as sources of crystalline seeds [15, 26–28]. A new method called \"Random Microseed Matrix Screening” and related techniques [29–32] that have been recently developed, involve crushing and preparing a seed-stock from microcrystalline material of any qual-ity present in one or more droplets of the initial crystallization screen. This method can also dispense nano-volumes of seed stock into all the conditions of the same or other screens. This procedure allows crystals to appear in screen conditions that are adequate for crystal growth, but not for nucleation. There is also a recently pub-lished new technique that combines the results of moderately suc-cessful initial screenings based on Genetic Algorithms [33].In order to initiate nucleation, nucleation-inducing particles or glass-based nucleants [34, 35], ultrasonic fields [36], or electromag-netic fields [37–46] have been applied, leading to conditions impos-sible to obtain by classical approaches. Subsequently, the growth of crystals can proceed by varying the temperature (either reducing or increasing it). Temperature can be modified to grow single crystals or to dissolve tiny crystals around a growing crystal. It is also possi-ble to avoid the formation of long, thin needles [22, 47] by moving to higher or lower temperatures. In the crystallization of proteins, temperature and mainly pressure have been poorly explored [22, 48–50]. There are usually two temperatures available (most com-monly 4 and 18 °C) for growing protein crystals. The existence of different polymorphs has been recently reported, after carefully test-ing a wide range of temperatures as well as other physicochemical parameters of the crystallization experiment [47, 51–54].Abel Moreno 55All concentrations should be measured in triplicate with an UV-VIS spectrophotometer, following the calibration procedures provided by the supplier. A calibration concentration plot can be obtained for each new protein, even if its extinction coefficient is not reported in the literature, for the calculation of protein concentration [55].Agarose gel 0.6% (w/v) stock solution of low melting point aga-rose (Tgel = 297–298 K, Hampton Research HR8-092) can be pre-pared by dissolving 0.06 g agarose in 10 mL of water heated at 363 K up to a transparent solution with constant stirring. The solution is passed through a 0.22 μm porosity membrane filter for removing all dust particles or insoluble fibers of agarose. The gel- solution can be stored in 1.0 mL aliquots in Eppendorf tubes in the refrigerator. Prior to crystallization in agarose gels, an Eppendorf tube of 1.0 mL is heated at 363 K in order to melt the gel. Most proteins are damaged when exposed to high tempera-ture, so it is best to mix only the precipitant agent with agarose to allow reaching the proper temperature without damaging the pro-tein. Although in the last decade agarose has been the most popu-lar gel for protein crystallization [56–58], there are other types of gels that have also been used for the same purpose [59–62].3 Advanced Crystallization Methods in PracticeAs mentioned in Subheading 2.1, it is important to separate the nucleation and crystal growth phenomena. This can be also accom-plished using electromagnetic fields. In particular, the use of electric fields has been shown to be useful for successful crystalliza-tion of proteins.For that purpose, one can use a crystal growth cell that consists of two polished float conductive ITO (Indium Tin Oxide Electrode) glass plates, 3.0 × 2.5 cm2, with a resistance ranging from 4 to 8 Ω (Delta Technologies, Minnesota, USA). The two electrodes are placed parallel to each other. The cell is prepared using a U-like or double well frame (for vapor diffusion set up) as shown in Fig. 3, made of elastic black rubber material, sealed with vacuum grease. Closure of the growth cell can be done by using a gun for melting silicone. The conductive ITO-coated surfaces are placed inwards, at 0.5 cm from each other, to provide appropriate connection area when applying direct (DC) or alternating current (AC) (Fig. 4a, b respectively). Each cell has a volume capacity of approximately 100 μL for precipitant (larger well) and 50 μL for protein plus precipitant (smaller well, as shown in Fig. 3 on the right), or a full volume of 200 μL when a batch configuration is used (Fig. 3, left). The sitting-drop vapor diffusion or batch crystallization conditions for each protein have to be properly established before applying the current. After closing the cell with a cover of melted silicone, 2.2 Protein Concentration2.3 Gel Preparation3.1 Experimental Setup for Constructing a Growth-Cell for Applying Electric FieldsAdvanced Protein Crystallization 56the system is connected to a DC source (Fig. 4a) that supplies direct current (ranging from 2 to 6 μA) or alternating current (ranging from 2 to 8 Hz), as shown in Fig. 4b. During nucleation, the AC or DC current is turned off after 48 h, so the nuclei are 0.15 cm3.0 cm 0.5 cm3.0 cmFig. 3 Different designs of e-crystallization growth-cell for applying electric field to the crystallization process of biological macromolecules. The two frames on the left are useful for batch crystallization setup, and the one on the right is for a vapor diffusion setupFig. 4 Two pieces of apparatus used for e-crystallization of proteins: (a) for applying DC ranging from 2–6 μA, (b) for applying AC during the crystallization of proteins. First, nucleation is induced and then the crystal growth proceeds via vapor diffusionAbel Moreno 57fixed on the surface of the ITO electrodes. After that the DC growth cell is left at a constant temperature to allow crystals to grow by vapor diffusion (Fig. 5a). In the case of AC, a current of 2 Hz will produce fewer crystals and at 8 Hz will produce a higher number of crystals, although smaller in size (Fig. 5b). Thus AC of 8 Hz or higher values could be used to prepare protein nanocrys-tals for XFEL experiments.New devices and novel methodologies to control nucleation and the size of crystals (utilizing glass beads for fragmentation of pro-tein crystals to be analyzed in a fine mesh grid via cryo-EM) have been recently described [5, 63]. Magnetic [64, 65] or electric fields [41, 66–68] have been applied in order to obtain larger and higher quality protein single crystals either for conventional X-ray crystal-lography or for neutron diffraction [69]. The use of AC currents has demonstrated that there is an effect on crystal size (see above). Higher frequencies (between 10 and 50 Hz) have produced tiny crystals for seeding purposes and for crystal growth research. 3.2 The Influence of Electric Fields in the Control of NucleationFig. 5 Crystals of glucose isomerase grown: (a) when a direct current (DC) of 2 μA is applied for 48 h and subsequently the crystal growth proceeds in 2 weeks by the sitting-drop setup, and (b) when an alternant current (AC) of 2 and 8 Hz is applied for 48 h. The bar scale for (b) is the same at 2 and 8 HzAdvanced Protein Crystallization 58There are other strategies that use specific electromagnetic fields to control transport phenomena [67, 70–73].In the particular case of ultrasonic and electric fields, one of the pioneering contributions to study the positive effect on the nucleation processes was the proposal by Nanev and Penkova [36] in 2001. The results of these experiments in which a 25 kHz ultra-sonic field (thermal double pulse technique) was applied to the crys-tallization process of lysozyme, demonstrated that the length of time required to obtain crystals, compared to the usual length, is reduced in half. However, the intensity of the ultrasonic field is a parameter to be considered, as the crystals broke mechanically, lead-ing to excess of nucleation and less time for the induction of grow-ing crystals. Along this line, other idea about using femtosecond lasers was developed in order to control nucleation [74–76]. Their use permitted to observe the area where the laser strike led to for-mation of only a few crystalline nuclei (this can be explained by the formation of small assemblies of protein that serve as seeds for grow-ing nucleation centers, produced by the focalized laser radiation).It has been shown that a growth-cell that utilizes electric fields (called e-crystallization cell with transparent electrodes), when applied to proteins, results in crystals that grow better oriented to the cathode (if the protein molecule was positively charged), com-pared to the crystals grown on the anode (negatively charged protein molecules) [42]. The batch method used to grow crystals applying either AC current [77–79] or DC [80–82] has been most widely used. However, in most cases, these batch crystallization conditions are not experimentally feasible to apply AC current to other proteins more than lysozyme [83]. A reengineered e- crystallization growth cell adapted to a sitting-drop setup has recently been described [42]. Another advantage has been reported for the experimental e-crystallization growth-cell, where after applying DC (to fix the nuclei on the electrode), the crystal growth process proceeds by vapor diffusion. Such a device has been used for crystallization and to search for different polymorphs of glu-cose isomerase [51] and lysozyme [70] at different temperatures. Along the crystal growth process, we usually obtain four different regimes: (1) induction/equilibration, (2) transient nucleation, (3) steady state nucleation and crystal growth, and (4) depletion [14]. During induction/equilibration, the sitting drop is equilibrating against the reservoir solution and becomes supersaturated when the electric field is applied; there were no nuclei visible in the light passing through the glasses of the ITO transparent electrodes. Eventually, no new crystals were formed and the existing protein crystal nuclei just continued to grow until completion of the pro-cess, reaching sizes from 100 to 300 μm, thus becoming suitable for X-ray crystallography. The crystals can be even used for diffrac-tion experiments in situ, if the commercially available ITO elec-trodes made of plastic material (polyethylene) are used.Abel Moreno 59A majority of the advanced methods mentioned in this chapter are based on the solubility diagram, such as that shown in Fig. 2a [11, 12, 21] or that phase diagram obtained from the physical and chemistry approaches [84]. Recently, advanced methods have been developed for obtaining very high quality crystals not only by growth in gels but also in the presence of strong magnetic fields. In the particular case of magnetic fields, whether they are homoge-neous or nonhomogeneous, they always act differently on samples. Nonhomogeneous magnetic fields are responsible for the reduc-tion of gravity forces on the solution through the action of the magnetic force [46, 64, 85]. By applying a vertical magnetic field gradient, a magnetizing force is generated on the sample. If this force is opposite to the gravitational force, the result will be a reduction in the vertical acceleration (effective gravity) with subse-quent decrease of natural convection [86]. Convection is practi-cally nullified, generating a situation similar to that found under microgravity conditions [45]. Furthermore, Wakayama et al. found that, in the presence of a magnetizing force opposite to \"g” (gravi-tational vector), fewer lysozyme crystals were obtained than in its absence [87]. The crystals that were obtained diffracted to a higher resolution, in agreement with the mathematical model [46].For experiments of protein crystallization under the influence of magnetic forces, all proteins and precipitating agents have to be mixed according to the known batch crystallization conditions. It is important to emphasize that the preparation of the batch solu-tion for crystallization must follow the rule that the most viscous solution must be added first, followed by the less viscous ones. Additionally, in order to guarantee highly ordered crystals, a gel can be introduced into the crystallization droplets. This must be done by mixing 1:1:1 (e.g., 5 μL + 5 μL + 5 μL) in the following order: precipitant, agar (0.60% w/v), and the protein. In the cases of standard solution, the gel might be replaced by water to preserve the same crystallization conditions as in the classic crystal growth methods. One must bear in mind that all concentrations from the stock solutions will be reduced to 1/3. Once mixed, the solution or the gelled mix is ready for the magnetic field experiments, as shown in Fig. 6. The mixture (prepared in 0.5 mL Eppendorf tubes) is drawn into a disposable 50 μL glass pipette of (Sigma-Aldrich Z-543292, 1.0-mm inner diameter), using capillarity forces. Green mounting clay from Hampton Research (HR4- 326) can be used to seal both ends of the capillary tubes. Once sealed, the capillary pipettes are introduced into an NMR glass tube (8 mm in diameter) and left for at least 48 h in the presence of a magnetic field generated in a 500–700 MHz (11.7–16.5 T) NMR instru-ment (Fig. 6). All experiments are performed at the temperature of the control unit of the NMR probe head, usually ranging from 291 to 293 K. The sample is left in the magnetic field for at least 2 days or more. Crystals will be better and larger if the time is longer. 3.3 Experimental Setup for Crystallization of Proteins Under the Influence of Magnetic FieldsAdvanced Protein Crystallization 60After the end of the experiment, the NMR tube is recovered from the magnet and the capillary pipettes are extracted from it. Then, the capillary pipettes are cut at both ends in order to harvest the crystals. The cut in the capillary pipettes can be done with a glass-capillary cutting stone (Hampton Research Cod. HR4-334). Once both ends of the capillary pipettes are opened, a little air pressure (applied by using plastic latex tubing attached to a 1 mL syringe for blowing it out, or by using a pipette bulb) is sufficient to expel the solution or gel with the crystals into a few microliters of a mother liquor or cryoprotectant on either a two- well or a nine-well glass plate. When necessary, the gel can be dissected with microtools in order to release the crystals. A small incision will open the gel and liberate the crystal to permit the cryoprotectant to enter and to replace the water molecules. All crystals should be immediately mounted and flash-cooled for X-ray data collection.We could observe better quality crystals when applying strong homogeneous magnetic fields, although the field effect was differ-ent depending on the space group in which the protein crystal-lized. The most remarkable effect of this strong magnetic force for the growth of lysozyme crystals was when they grew in the polar space group P21 [65]. The viscosity of the solution increased when magnetic fields of 10 T were applied [88, 89]. The increase in vis-cosity was translated into reduced convection. In addition, an 3.4 The Influence of Magnetic Force to Orient and to Grow Large Protein CrystalsFig. 6 A setup for experiments performed in the presence of strong magnetic field. Two types of capillaries are used: glass pipettes and NMR tubes. The magnetic field should be applied from 500 to 700 MHz (11.7–16.5 T) by using an NMR apparatus, this is that commonly used in analytical chemistry laboratoriesAbel Moreno 61orientation effect was observed in the crystals formed under high magnetic fields. In a more recent study, decreasing the diffusion coefficient of lysozyme was assessed in a crystallization solution exposed to a homogeneous magnetic field of 10 T [44, 65, 90]. All these observations are interrelated and are due to the orienting effect of the magnetic field at a microscopic level. In a supersatu-rated solution, protein nuclei are in suspension in the solution and sediment when reaching an adequate size, which depends on the value of the field. These nuclei would act as blocks that hinder free diffusion of monomers, making the solution more viscous and, hence, lowering convection. Additionally, paramagnetic salts will produce multiple orientation responses to the application of strong magnetic fields [91].Figure 7 shows the results of growing lysozyme and glucose isomerase crystals for 1 week inside a 700 MHz (16.5 Tesla) NMR magnet. To achieve this, it is necessary to know the conditions of batch crystallization of the protein under study. Once these condi-tions are known, the time needed to induce nucleation must be known and, finally, access to an NMR equipment of at least 500 MHz (11.7 MHz) or higher is needed to grow large protein crystals. The equipment must be available for the duration of the experiment (at least 3 consecutive days).Fig. 7 Crystals of glucose isomerase: Control (a) and (b) grown in the presence of a magnetic field of 700 MHz (16.5 T). Crystals of lysozyme used as control (c), and (d) grown in the presence of a magnetic field of 700 MHz (16.5 T). The control crystals are four times smaller than those obtained inside the magnetAdvanced Protein Crystallization 62Studies of the influence of magnetic fields on crystal growth have been conducted during the last 15 years and they are still being continued [44, 46, 87]. There is still much to be learned about the effect of homogeneous and nonhomogeneous magnetic fields in solutions on a variety of biological macromolecules [89, 92, 93]. All these phenomena apparently favor the quality of the resulting crystals, although we still need more detailed research to understand the underlying mechanisms [64, 94]. There have been a few efforts in this respect, such as combining the positive effect of crystal growth in gels and strong magnetic fields to prove that the crystal growth kinetics is quite close to that obtained in micro-gravity conditions [43, 65, 86]. The effects of many physical parameters, such as electrical [66, 67, 77, 78] and magnetic fields [45, 46, 64, 65] on the control of nucleation and growth of pro-tein crystals have been assessed. On the other hand, combining the electric and magnetic fields in order to influence crystal orientation can also benefit its homogeneous size in average of many crystals at the same time [68]. One of the main advantages of growing crys-tals under magnetic fields for a long time (1–3 weeks) is the ability to control their size. The large crystals obtained by applying mag-netic force could be suitable not only for neutron diffraction exper-iments, but also for conventional X-ray crystallography, since one large crystal could yield several data sets of high quality.Recently, several reviews demonstrated the potential of growing protein crystals in gels, which produce crystals of high quality for high-resolution X-ray crystallography compared to the crystals obtained in solution (Fig. 8a) [25, 59, 62, 95, 96]. Another way of reducing the natural convection of solutions under earth gravity is to incorporate jellified media into the solutions. Already in 1968, Zeppezauer et al. [97] described the use of micro-dialysis cells formed by capillary tubes sealed with gel caps (polyacrylamide) for reducing convection in crystallization solutions, and obtaining bet-ter crystals. In 1972, Salemme also applied crystallization inside a glass capillary tube [98], placing a protein solution in contact with the precipitating agent solution and reaching equilibrium through counter-diffusion. That technique was subsequently used to crystal-lize the ribosomal subunits [99]. The combination of gel- growth and the use of capillary tubes have led to the production of a con-siderable number of protein crystals by counter-diffusion methods [25, 95]. The historical journey of counter-diffusion methods and its fundamentals and experimental development are described below. These counter-diffusion techniques, based on diffusion-con-trol transport processes [100–104], can also be considered as advanced methods for protein crystallization. The gel- growth tech-nique has been used for the crystallization of inorganic salts and it was first applied for protein crystallization at the beginning of 1990s [105–107]. The counter-diffusion methods have proved efficient 3.5 Crystallization by Counter-DiffusionAbel Moreno 63and effective in crystallizing a certain number of proteins, which could not be crystallized by conventional approaches [25, 95, 96].García-Ruiz and Henisch theoretically proposed in the middle of 1980s the use jellified media to crystallize biological macromol-ecules by the gel-growth method (Fig. 8b, c) [102, 103]. This technique, based on the principles of reduced convection and dif-fusion transport, also offers an advantage of including a wide range of consecutive conditions in a single experiment [108, 109]. These advances permitted García-Ruiz and his team to develop, in 1993, the first variant of the counter-diffusion methods, called the gel acupuncture technique (Fig. 9a) [24]. This novel technique uti-lizes a precipitating agent that diffuses through the gel support by the capillary force inside a capillary tube filled with a protein solution, thus enabling crystallization [110]. Nowadays, this tech-nique is better known thanks to the assessment of the different types of gels, capillaries, additives, as well as the type of precipitat-ing agents that can be used [24, 59, 62, 111, 112]. In contrast to other techniques that use capillaries, different levels of supersatura-tion can exist, allowing precipitation to occur in very high super-saturation zones (nucleation occurs when supersaturation is high, and the growth of the nuclei when supersaturation diminishes), increasing the probability of finding adequate crystallization con-ditions [100, 108]. Another advantage includes a possibility of crystallizing proteins in capillaries smaller than 0.5 mm in diameter Fig. 8 Crystals of the enzyme aspartyl t-RNA synthetase grown: (a) in solution, (b) in an agarose gel (0.2% w/v), and (c) in a silica gel obtained by the neutralization of sodium metasilicate. As reference, the size of the larger crystal in (a) is 100 μm. In (b) both crystals are 200–250 μm, and in (c) ~400 μmAdvanced Protein Crystallization 64(sometimes this allows to obtain cylinders of protein crystals). This helps to avoid their later physical manipulation and the risk of breakage when collecting X-ray diffraction data [56, 113]. Additionally, this method allows crystallization of macromolecules in the presence of cryoprotectant agents and/or heavy metals.By means of crystallization strategies utilizing the counter- diffusion method (Fig. 9b) it has been possible to crystallize a vari-ety of proteins with different molecular weights and with a wide range of isoelectric points, as well as viruses and protein-nucleic acid complexes [25, 95, 96]. In addition, as demanded by the advances in structural proteomics, there is a device that allows mul-tiple simultaneous and independent crystallization assays suitable for an effective screening of crystallization conditions [113]. It combines the advantages of multiple conditions inside a capillary, increasing the chances of finding the optimal conditions and the possibility to obtain diffraction data directly from the crystalliza-tion device. This would turn this method into the first fully auto-mated process, leading from the initial stages until data acquisition for structural analysis.Precipitating agentPrecipitating agentabHydrogelProtein+AgaroseProtein solutionPlugs ofplasticinesealed withnail varnishfor closingthe capillarytubeFig. 9 Tw o b as ic exp er im en ta l s et up s of the cou nt er- di ff us io n meth od s. (a) The gel acupuncture method (known as GAME) is shown on the left. The capillary tubes are inserted into the gel, the protein is inside the capillaries and the precipitant on top of the gel. (b) The right panel illustrates counter diffusion in Lindemann capillary tubes, where the protein is mixed with agarose (or any other gel) and the precipitant is applied on top of the gelAbel Moreno 654 Other Practical ApproachesYears of experimenting with different crystals have confirmed that by minimizing the convective transport of mass, it is generally pos-sible to obtain higher quality crystals, with improved mechanical and optical properties, with reduced density of defects, and larger in size.It is natural to think that reduction or absence of gravity will lead to superior quality crystals [114–117]. Experimental observa-tions and data support the hypothesis that convective flow intro-duces statistical disorder, defects, and surface dislocations into growing crystals [118–123]. Convective transport tends to be vari-able and erratic, generates variations in supersaturation levels around the crystal faces that are being formed, and exposes them to permanently high nutrient concentrations, equal to those inside the solution. However, in microgravity, where convection is sup-pressed, a reduction in nutrients concentration is produced in the crystal interphase. Transport is then purely diffusive, which is very slow for proteins. This gives rise to a \"nutrients diminution zone” around the nucleus and, due to the absence of gravity, the nucleus is quasi-stable. Generally speaking, the nutrient molecules diffuse very slowly due to their size, which lengthens and extends the nucleation. Large aggregates diffuse even more slowly than the monomers that form the crystal. Hence, the vacuum zone acts as a \"diffusive filter,” preventing their incorporation into the growing crystal. Apparently, this is the main mechanism responsible for the improvement in the crystal quality due to microgravity. This hypothesis is not only supported by experimentation but also by a mathematical model that explains the transport process [124].Microgravity experiments, in which the lack of convection leads to impressive results, have been evolving. It is now possible to perform a multitude of simultaneous experiments. However, there are still two criteria that can be applied for optimizing crystal-lization conditions: namely (1) one performs several assays that assess a wide range of conditions, consuming a large amount of material, or (2) one adjusts beforehand the preliminary conditions for future space missions. However, the consecutive missions may be delayed for months or years, which will counter the advantages of the microgravity method [125].Utilization of microgravity has been reborn at the beginning of the twenty-first century, but only for crystallization of macromo-lecular complexes that have never been observed in crystalline form on Earth [126–128]. It is not surprising that most of the effort put in this new trend will offer interesting results that were difficult to get in the past due to uncontrolled experimental conditions in the rockets or during space missions (temperature variations, pressure issues, inadequate containers, etc.). We should expect specific 4.1 Crystallization in MicrogravityAdvanced Protein Crystallization 66missions for specific problems in protein crystallization in the near future (perhaps the intrinsically disordered proteins would give us some structures that are hard to obtain on Earth or never seen in a crystalline state) [129, 130].When we do not know the crystallization conditions for a protein, bioinformatics analysis to predict if the protein is not intrinsically disordered should be performed first. Next, one should first make simple solubility tests (precipitation with ammonium sulfate (AMS), polyethylene glycols (PEGs), to try different temperatures for crystallization experiments as well as different pH values). Nowadays, there are commercial kits available; these are tools that allow investigating many crystallization conditions based on statis-tical analysis of protein crystallization. They are based on sets of conditions published at the beginning of the 1990s and even more recently [131, 132]. Crystallization robots have been developed to facilitate screening of hundreds of conditions in a short time. Once an adequate crystallization condition has been found, it can be refined by screening around it. However, there is an additional limitation if the protein under investigation is intrinsically disor-dered. Many proteins require ligands to stabilize their fold and to allow them to crystallize more readily. The main characteristics of the strategies and limitations of how to stabilize a protein were reviewed elsewhere [133, 134]. It was shown that 100 out of 200 proteins had been crystallized thanks to the use of specific ligands, although not all of them crystallized favorably. The use of amino acids and their analogs has been widely studied and yielded prom-ising results [134]. Details on how to use specific ligands and nucleants in order to crystallize any protein were reviewed else-where [135, 136]. Pharmaceutical companies have used these strategies to investigate drugs targeted for diverse diseases. The system MAESTRO (http://www.schrodinger.com) is a suite of programs based on computational chemistry, enabling the predic-tion of the most probable molecules and bonds that can be used to stabilize protein, RNA, DNA, or macromolecular complexes.The limited availability of many proteins is often the key impedi-ment in crystallographic research [137], emphasizing the need for systems that require minimal amounts of protein for crystalliza-tion. This is easy when working on the scale of liters or milliliters, but the process gets complicated as we lower the scale by 5 or 6 orders of magnitude [138]. In this way, devices that use microflu-idics arise as potential tools for protein crystallization due to their ability to perform many experiments in reduced volume.Among the desired features of these \"microfluidic chips,” we can highlight injection of very exact solution volumes and high reproducibility of the results [139–141]. They are characterized by either a low Reynold’s number, or a lack of turbulence, which 4.2 The Use of Ligands to Stabilize and Crystallize Proteins4.3 Application of Microfluidics to Protein CrystallizationAbel Moreno 67allows only laminar flows, and ultra-fast diffusive mixtures [142]. Due to a density gradient, the microfluidic systems present either a low Grashof number or the absence of convection. This property demonstrates that it is possible to crystallize proteins with very effective kinetics [143, 144]. In the work of Hansen et al., [143] many parallel reactions were performed. The necessary solutions were introduced either manually or with the help of a robot, into 48 wells. The protein and the precipitating agents were placed in individual chambers that were later connected by eliminating the separating barrier. The total volume of the two chambers was 25 nL, and the relations between both species were set when designing the chip (in this case, they were 1:4, 1:1, and 4:1). Thanks to this device, 11 different macromolecules were success-fully crystallized and one was used for diffraction experiments. Among the advantages of these devices [143] are the very precise measurement of the amount of solutions, the absence of the effects of viscosity that affect diffusion of molecules, the ease of harvesting the grown crystals, and the fact that liquid-liquid diffusion meth-ods can be applied in the presence of gravity due to the absence of convection. With the use of microfluidics, equilibrium is achieved faster and the time to grow crystals is reduced. The plan for the future is to enable time-resolved serial crystallography using smaller size chips suitable for collecting X-ray data in situ [139].However, the microfluidic chips still pose some disadvantages that must be addressed before they can be implemented for large- scale crystallization. Among the disadvantages is the permeability of the elastic connections. Another disadvantage is that it is hard to implement optimization stages, as the experiment starts with pre-mixed solutions (stocks). In the future, it would be advisable to incorporate a chip of this type that can prepare solutions and to cou-ple it in a series [145, 146]. On the other hand, harvesting of crystals is a manual process, in which the whole device is opened, increasing the risk of losing the remaining crystals in order to extract just one.The design of these devices has been possible thanks to advances in engineering; however, the cost is still very high com-pared to the traditional systems. Fabrication of chips, which are similar to integrated circuits, requires strict control of cleanliness in the process because micrometric lines are being manufactured. The equipment used for their manipulation is usually very sophis-ticated and costly and can be used for just one experiment. The advantages of microfluidics have been recently demonstrated for different applications using graphene and a variety of materials in the fabrication of the chips, even when applied for the crystalliza-tion of membrane proteins [142, 147–157].Recent advances in genomics have led to large-scale efforts in structural biology in a variety of biological samples [157], culmi-nating in Structural Proteomics Consortia and in granting large 4.4 Automation of Mass Crystallization (High-Throughput)Advanced Protein Crystallization 68public subsidies to scientific laboratories as well as to private enter-prises, particularly pharmaceutical companies [158, 159]. Projects on metabolomics are diverse and range from studies of structure–function relationships, through mechanisms involved in protein folding and applications to biomedical research [160], to a more pragmatic focus, involving rational design of drugs based on the structure of their target molecules. The use of X-ray crystallogra-phy is critical in these studies [161].Laboratories specializing in structural biology have, in theory, the capacity for handling large-scale projects that require maximal automation at all stages, including crystallographic research [5, 162, 163]. This is not a big issue, particularly if we consider crys-tallization through microfluidic techniques. In fact, there are already different types of robots on the market that perform these functions. For example, Decode Biostructures produces ROBOHTC, comprising a robot that prepares different crystalli-zation solutions (Matrix Maker) and another robot that arranges the drops. Douglas Instruments is responsible for ORYX 6, which can be used to perform vapor diffusion assays through sitting drops, as well as microbatch assays. This company has also devel-oped a random micro-seeding matrix screening for high through-put hints for protein crystallization conditions [30]. This robot can set up 240 cells per hour. Another commonly used robot is the TTP LabTech Mosquito. This robot contains a set of precision micropipettes mounted on a continuous band, which dispense drop volumes from 50 nL to 1.2 μL. Discarding the disposable micropipettes avoids cross-contamination and eliminates exhaus-tive washings time. The equipment permits sitting drop or micro-batch crystallization tests, as well as hanging drop experiments. It can dispense drops into plates with 96, 384, or 1536 wells.Up to now we have generally mentioned the advances and drawbacks of large-scale structural biology. Although most experi-ments have dealt with soluble proteins rather than membrane pro-teins, high-throughput methodologies have nonetheless been implemented also for membrane protein crystallization [149, 153, 164]. Many strategies and techniques known as in meso crystalli-zation [165, 166] (including crystallization in lipid cubic and sponge phases) have allowed the determination of several hundred membrane protein structures [167, 168].Finally, we can understand that the advances in the processes and automation made in the past have allowed structural biology to be developed worldwide [169]. Many laboratories are able to successfully clone, express, purify, and crystallize soluble proteins at a rate that was unthinkable some years ago. However, there is still much to be done to control and predict each of the different stages of the general process. A summary of all types of possibilities provided by the high-throughput equipment related to proteins crystallization, has been reviewed and published elsewhere [170].Abel Moreno 695 Criteria to Analyze Crystal QualityBeautiful crystals do not necessarily diffract X-rays to high resolu-tion, but only a few publications have dealt with the strategies for increasing crystal quality [171]. A majority of publications were focused on proteins, though the principle can be applied to other biological macromolecules (DNA, RNA, polysaccharides, macro-molecular complexes) [136, 172, 173].The most adequate techniques to estimate the quality of a crystal are those that employ X-ray topography [174–178]. Here the diffraction equipment is placed in a very characteristic way and the crystal oriented in a preferred direction. Once this has been achieved, the diffraction of a spot is followed through the Ewald sphere (around the crystal), and its quality is characterized through rocking curves. The obtained curves are processed with specific programs that allow us to determine the crystal quality very accu-rately. If the curve is very fine or pointed, we can confirm that the quality of the crystal is very good; and on the contrary, when the curve is Gaussian-shaped, we can confirm that the quality of the crystal is not very good.All the advanced methods for protein crystallization mentioned here are the result of the developments in biological crystallogen-esis. The name \"protein crystallogenesis” was coined by Richard Giegé in the middle of 1990s [179]. It is an outstanding science that studies all the physicochemical processes that govern the growth of crystals of biological macromolecules [180]. The meth-ods, strategies, and devices used to obtain high quality crystals for X-ray crystallography are also part of this fascinating science.AcknowledgmentsThe author acknowledges the support from the DGAPA-UNAM Project PAPIIT No. IT200215. 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J Cryst Growth 196: 559–571 178. Otalora F, Garcia-Ruiz JM, Gavira JA et al (1999) Topography and high resolution dif-fraction studies in tetragonal lysozyme. J Cryst Growth 196:546–558 179. Giege R, Lorber B, Theobald-Dietrich A (1994) Crystallogenesis of biological macro-molecules—facts and perspectives. Acta Crystallogr D Biol Crystallogr 50:339–350 180. Gavira JA (2016) Current trends in protein crystallization. Arch Biochem Biophys 101: 3–11Abel MorenoCitations (8)References (190)... The principle of the method benefiting from electric fields relies on the separation of the nucleation and crystal growth; it can be applied in the electric as well as the electromagnetic field. The nucleation process takes place while the current is switched off [28]. In order to perform crystallization experiments in the electric field, the specially designed crystal growth cell has to be used. ...... It was reported that the current positively influences the size of protein crystals. Specifically, lower frequencies of alternating current provide less larger crystals compared to higher frequencies [28]. For the purposes of using electric current the e-crystallization cells based on vapour diffusion or batch method were made [29]. ...... Moreno 2017 mentions the use of gels for obtaining large well-arranged crystals together with use of magnetic forces for at best two days and more days. For this type of experiment, the capillary glass pipettes were reported to be suitable for use of homogeneous or nonhomogeneous magnetic fields [28]. The best influence of magnetic field use is regulation of quality and crystal size [32]. ...Advanced BiocrystallogenesisChapterFull-text availableApr 2021 Ivana Kuta Smatanova Petra Havlickova Barbora Kaščáková Tatyana PrudnikovaNowadays, X-ray crystallography is one of the most popular structural biology methods. Successful crystallization depends not only on the quality of the protein sample, precipitant composition, pH or other biophysical and biochemical parameters, but also largely on the use of crystallization technique. Some proteins are difficult to be crystallized using basic crystallization methods; therefore, several advanced methods for macromolecular crystallization have been developed. This chapter briefly reviews the most promising advanced crystallization techniques and strategies as one of the efficient tools for crystallization of macromolecules. Crystallization in capillaries, gels, microfluidic chips, electric and magnetic fields as well as crystallization under microgravity condition and crystallization in living cells are briefly described.ViewShow abstract... Traditionally, most of the experimental studies were performed with hen-egg white lysozyme (HEWL). Three reviews [Al-haq, 2007, Frontana-Uribe, 2008, Hammadi, 2009b, and a book chapter [Moreno, 2017] have already been published. ...... Table Appen Crystallisation could occurs via external physical factors, such as magnetic and electric fields (EFs); proper crystallisation conditions can be fine-tuned using variation of both direct current (dc) [Adrjanowicz, 2018] and alternating current (ac) EFs. Three reviews [Al-haq, 2007, Frontana-Uribe, 2008, Hammadi, 2009b, and a book chapter [Moreno, 2017] have already been published on that subject. ...Laser-Induced Nucleation in a Coaxial Microfluidic MixerThesisJun 2019Zhengyu ZhangCrystallization is one of the elementary operations of chemical engineering. The materials produced are extracted by crystallization and purified by recrystallization. But the nucleation of the crystal remains a mystery and the classical theory of nucleation is undermined by numerous experimental data. We have chosen to build a microfluidic precipitation device by mixing solvents to produce and continuously observe the birth of a large number of crystals. The molecule chosen for the study is DBDCS, with fluorescent crystals but not the molecule. The germ will thus be the first luminous object in the mixture.We calculated the thermodynamics of the ternary mixture DBDCS/diox/water from what is known for the mixture diox/water and the solubility curve of DBDCS in diox/water, as part of a regular solution model. We calculate the conditions of the spinodal decomposition ([DBDCS]= 59 times the saturation) of the ternary mixture into a hypothetical liquid phase of DBDCS practically pure in a diox/water mixture.However, this hypothetical phase we observe it as the main initial product of this precipitation experience. The measurement of the volume produced by this liquid phase confirms that it is practically pure. The appearance of this liquid phase requires a strong oversaturation following the diffusion of water. The study of the solubility of DBDCS in diox/water shows that the chemical potential of DBDCS in water is 17 RT higher than its value in diox. The diffusion of water in diox induces the formation of an energy barrier that repels and concentrates DBDCS to the center of the device. The study of the time taken to reach the critical concentration as a function of the initial concentration of DBDCS in the central flow provides a value 50 to 70 times the saturation for the critical concentration of occurrence of the DBDCS liquid phase. This confirms that we observe a spinodal decomposition. The product of this spinodal decomposition is a cloud of sub-micrometric droplets. But the chemical potential gradient can, under certain conditions, group these nanodrops into a string of micrometric drops of the same size.When the anti-solvent is not pure water, but a diox/water mixture, the potential barrier does not outweigh the entropy of the diffusion. This is shown by the distribution of the fluorescence of the molecules (yield 10-4). Over times of the order of 5s, the formation and growth of crystals is observed. The numerical simulation indicates that under the conditions the relative oversaturation does not exceed 3.5. Rapid imaging and fluorescence allow the crystals to be observed one by one. Three different polymorphs are identifiable by their lifetime : the green and blue phases already observed and a short-lived phase. The growth rates are widely dispersed, making it difficult to locate and observe spontaneous nucleation.By focusing a laser on the clouds of nanodrops, we observe an optical tweezer effect capable of collecting these drops. By focusing this laser in the zone of maximum super-saturation under spontaneous nucleation conditions, we observe a multiplication of the number of crystals formed by a factor of five. We are in the presence of laser-induced nucleation. These crystals have the same growth rate, the same distribution in number of polymorphs, as the spontaneously obtained crystals. This laser-induced nucleation is therefore very soft and induces a minimal change in the nucleation mechanism. An optical tweezer effect that locally concentrates the precursors of the germ and increases the over-saturation could have this effect.This laser-induced nucleation makes it possible to locate the nucleation. At the focal point of the NPLIN laser, we observe the accumulation of a phase with a short fluorescence lifetime, so it can be disordered, which disappears after the passage in the laser while the green phase grows slowly. This would be a direct observation of a two-step nucleation.ViewShow abstract... Traditionally, most of the experimental studies were performed with hen-egg white lysozyme (HEWL). Three reviews [Al-haq, 2007, Frontana-Uribe, 2008, Hammadi, 2009b, and a book chapter [Moreno, 2017] have already been published. ...... Table Appen Crystallisation could occurs via external physical factors, such as magnetic and electric fields (EFs); proper crystallisation conditions can be fine-tuned using variation of both direct current (dc) [Adrjanowicz, 2018] and alternating current (ac) EFs. Three reviews [Al-haq, 2007, Frontana-Uribe, 2008, Hammadi, 2009b, and a book chapter [Moreno, 2017] have already been published on that subject. ...Curriculum research on Sustainable Development Education in Chinese Higher Education -- Education for SD, SD for EducationThesisNov 2020Tongzhen ZhuMy dissertation is on the subject of \" Sustainable Development education in higher education system”. It s a sociology and didactic research on the curriculum of the major on Sustainable Development in higher education.Education on Sustainable Development (ESD) has been rapidly increasing in popularity. Its importance was realized when the United Nations Decade for Education for Sustainable Development (UNDESD) was established in 2004 by UNESCO. Education on Sustainable Development can be termed as altering ways of thinking. This is where the resources used for education are utilized such that the generations to come can access the items of Sustainable Development. It is a process that is educational that is categorized by methods and approaches focused on establishing awareness on issues related to sustainable development. This is different from the approaches that have been there in the past where importance was placed on issues that were environmental. The scope of education on sustainable development is wider (I will discuss the differences between the education on sustainable development and the education on environment) whereby it has placed focus on processes, means and tools that give people the opportunity to form knowledge, skills, competencies and values that are required when it comes to contributing towards society that is more sustainable.Educators worldwide have been influenced to change their contents of teaching so that the education systems can be able to respond better to challenges that are socio-economic globally, regionally and locally. Moreover, new emphasis have been seen when it comes to innovative teaching development methods in relation to sustainable development. This was according to the report that was done by UNESCO that placed emphasis on learning and education on sustainable development context; it also included the stakeholders involved in all levels as well as all the regions included in the UN. At the level of theoretic research, the economic domain, the ecology domain and the social domain still the three mainstream areas.In recent years, institutions of education worldwide have been advised to encourage the students by mobilizing them and also participate more actively in global, national and local processes when it comes to sustainable or durable development (Barth, 2015). In the past three decades (I counted from 1987 ,Brundtland report ), sustainability was not well understood, but recently more and more researches and jobs booming, offering us more clear definition and approaches to work. A lot of opportunities exist when it comes to learning on sustainable, also learning about sustainability and at the same time a lot can be gained from it. Some of the education sectors that have been affected strongly by Education on Sustainable Development especially the higher education which is seen to have an important role when it comes to the debate on Education on Sustainable Development. Because of the strategic and positioned nature of institutions of higher education, credible evidence shows that they should and can make contributions that are strong when it comes to sustainable development in general. In addition, institutions for higher learning have to implement the sustainable development education specifically.ViewShow abstract... This integration of microscopy fabrics and textures with diagenetic phase transitions (illustrated by the blue text and images) establishes a new classification scheme for stones that is independent of the location of their genesis within the kidney. This re-synthesis reflects the multiple pathways of crystallization and dissolution in natural systems as defined by ΔG and diagenetic phase transitions presented 30,37,38,52,81,140,141,146,148,149,155,158,161,205,206,212,[294][295][296][297][298][299][300] . b | Changes in ΔG during sequential diagenetic phase transitions in phosphate, carbonate and silicate systems in comparison with human kidney stone formation. ...... b | Changes in ΔG during sequential diagenetic phase transitions in phosphate, carbonate and silicate systems in comparison with human kidney stone formation. This re-synthesis incorporates our understandings of ΔG pathways and diagenetic phase transitions observed in nature 30,37,38,52,81,140,141,146,148,149,155,158,161,205,206,212,[294][295][296][297][298][299][300] . Note that silicate reactions can also be involved in carbonate diagenetic phase transitions 299,300 . ...Human kidney stones: a natural record of universal biomineralizationArticleFull-text availableMay 2021NAT REV UROL Mayandi SIVAGURU Jessica J. SawElena M. Wilson Bruce FoukeGeoBioMed — a new transdisciplinary approach that integrates the fields of geology, biology and medicine — reveals that kidney stones composed of calcium-rich minerals precipitate from a continuum of repeated events of crystallization, dissolution and recrystallization that result from the same fundamental natural processes that have governed billions of years of biomineralization on Earth. This contextual change in our understanding of renal stone formation opens fundamentally new avenues of human kidney stone investigation that include analyses of crystalline structure and stratigraphy, diagenetic phase transitions, and paragenetic sequences across broad length scales from hundreds of nanometres to centimetres (five Powers of 10). This paradigm shift has also enabled the development of a new kidney stone classification scheme according to thermodynamic energetics and crystalline architecture. Evidence suggests that ≥50% of the total volume of individual stones have undergone repeated in vivo dissolution and recrystallization. Amorphous calcium phosphate and hydroxyapatite spherules coalesce to form planar concentric zoning and sector zones that indicate disequilibrium precipitation. In addition, calcium oxalate dihydrate and calcium oxalate monohydrate crystal aggregates exhibit high-frequency organic-matter-rich and mineral-rich nanolayering that is orders of magnitude higher than layering observed in analogous coral reef, Roman aqueduct, cave, deep subsurface and hot-spring deposits. This higher frequency nanolayering represents the unique microenvironment of the kidney in which potent crystallization promoters and inhibitors are working in opposition. These GeoBioMed insights identify previously unexplored strategies for development and testing of new clinical therapies for the prevention and treatment of kidney stones.ViewShow abstract... Initial crystallization screening of TvCyP2 involved using commercial crystallization screen kits (Hampton Research, Jena Bioscience, and Qiagen, Aliso Viejo, CA, USA), 96-well Intelli-plates (Art Robbins Instruments, Sunnyvale, CA, USA) and a Phoenix robot (Art Robbins Instruments, Sunnyvale, CA, USA) at 298 K. The sitting drop vapor diffusion method [38,39] was used to grow crystals. The 0.6-3 µm crystals of TvCyP2 appeared within 5 days in the commercial kit conditions. ...N-Terminal Segment of TvCyP2 Cyclophilin from Trichomonas vaginalis Is Involved in Self-Association, Membrane Interaction, and Subcellular LocalizationArticleFull-text availableAug 2020 Sarita AryalHong-Ming Hsu Yuan-Chao LouChinpan ChenIn Trichomonas vaginalis (T. vaginalis), cyclophilins play a vital role in dislodging Myb proteins from the membrane compartment and leading them to nuclear translocation. We previously reported that TvCyP1 cyclophilin from T. vaginalis forms a dimer and plays an essential role in moving the Myb1 transcription factor toward the nucleus. In comparison, TvCyP2 containing an extended segment at the N-terminus (N-terminal segment) formed a monomer and showed a different role in regulating protein trafficking. Four X-ray structures of TvCyP2 were determined under various conditions, all showing the N-terminal segment interacting with the active site of a neighboring TvCyP2, an unusual interaction. NMR study revealed that this particular interaction exists in solution as well and also the N-terminal segment seems to interact with the membrane. In vivo study of TvCyP2 and TvCyP2-∆N (TvCyP2 without the N-terminal segment) indicated that both proteins have different subcellular localization. Together, the structural and functional characteristics at the N-terminal segment offer valuable information for insights into the mechanism of how TvCyP2 regulates protein trafficking, which may be applied in drug development to prevent pathogenesis and disease progression in T. vaginalis infection.ViewShow abstract... Traditional crystallization methods can be classified into the three basic categories of batch diffusion, vapor diffusion and liquid diffusion, which have been developed and utilized with HTPCS technologies [16]. In recent years, new crystallization methods such as in vivo protein crystallization, counter-diffusion technique, supercritical fluid crystallization, electric field, magnetic field, microgravity controlled precipitation, agarose gel solution crystallization can be used to produce nanocrystals [17]. The most remarkable improvement is being done on the microfluidic array chip with micrometer-sized crystallization space [18]. ...What’s happened over the last five years with high-throughput protein crystallization screening?ArticleFull-text availableApr 2018Expet Opin Drug Discov Frank LinViewCyclodextrin and its derivatives enhance protein crystallization by grafted on crystallization platesArticleMar 2020J CRYST GROWTHWang Qian-JinZhao Gang Chenyan ZhangProtein drugs are attracting increasing attention. Eight of best-selling drugs in worldwide are proteins. Crystallization is an important protein purification method; however, it remains a bottleneck step. Heterogeneous nucleate is an effective method of enhancing protein crystallization. Cyclodextrin has been widely used in pharmaceutics, and our previous study showed that it is an effective heterogeneous nucleate for enhancing protein crystallization, and it may be widely used in purification of protein drugs. However, cyclodextrin must be added into the crystallization plate wells by hand, which is labor intensive and is not beneficial to automated crystallization screening. We grafted β-cyclodextrin (β-CD) and its derivatives (i.e., p-toluenesulfonyl-β-cyclodextrin [PTCD], mono-(6-(1,6-hexamethylenediamine)-6-deoxy)-β-cyclodextrin [MHCD] and mercapto-β-cyclodextrin [MCD]) on crystallization plates and tested their effects on protein crystallization. Protein crystallization success rate was improved, particularly for the PTCD-grafted group. This is an easy method to facilitate protein crystallization and can be widely applied for automatic crystallization screening.ViewShow abstractStructural Studies of Autophagy-Related Proteins: Methods and ProtocolsChapterJan 2019Meth Mol Biol Melanie SchwartenOliver H Weiergräber Dusan PetrovicDieter WillboldInformation about the structure and dynamics of proteins is crucial for understanding their physiological functions as well as for the development of strategies to modulate these activities. In this chapter we will describe the work packages required to determine the three-dimensional structures of proteins involved in autophagy by using X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. Further we will provide instructions how to perform a molecular dynamics (MD) simulation using GABARAP as example protein.ViewShow abstractProtein Crystallization in an Actuated Microfluidic Nanowell DeviceArticleFull-text availableMar 2016CRYST GROWTH DES Bahige Abdallah Shatabdi Roy-Chowdhury Raimund Fromme Alexandra RosProtein crystallization is a major bottleneck of structure determination by X-ray crystallography, hampering the process by years in some cases. Numerous matrix screening trials using significant amounts of protein are often applied, while a systematic approach with phase diagram determination is prohibited for many proteins that can only be expressed in small amounts. Here, we demonstrate a microfluidic nanowell device implementing protein crystallization and phase diagram screening using nanoscale volumes of protein solution per trial. The device is made with cost-effective materials and is completely automated for efficient and economical experimentation. In the developed device, 170 trials can be realized with unique concentrations of protein and precipitant established by gradient generation and isolated by elastomeric valving for crystallization incubation. Moreover, this device can be further downscaled to smaller nanowell volumes and larger scale integration. The device was calibrated using a fluorescent dye and compared to a numerical model where concentrations of each trial can be quantified to establish crystallization phase diagrams. Using this device, we successfully crystallized lysozyme and C-phycocyanin, as visualized by compatible crystal imaging techniques such as bright-field microscopy, UV fluorescence, and second-order nonlinear imaging of chiral crystals. Concentrations yielding observed crystal formation were quantified and used to determine regions of the crystallization phase space for both proteins. Low sample consumption and compatibility with a variety of proteins and imaging techniques make this device a powerful tool for systematic crystallization studies.ViewShow abstractThe Stanford Automated Mounter: Pushing the limits of sample exchange at the SSRL macromolecular crystallography beamlinesArticleFull-text availableApr 2016J Appl Crystallogr Silvia RussiJinhu SongScott E. McPhillips Aina E CohenThe Stanford Automated Mounter System, a system for mounting and dismounting cryo-cooled crystals, has been upgraded to increase the throughput of samples on the macromolecular crystallography beamlines at the Stanford Synchrotron Radiation Lightsource. This upgrade speeds up robot maneuvers, reduces the heating/drying cycles, pre-fetches samples and adds an air-knife to remove frost from the gripper arms. Sample pin exchange during automated crystal quality screening now takes about 25 s, five times faster than before this upgrade.ViewShow abstractThe role of mass transport in protein crystallizationArticleFull-text availableFeb 2016 Juan Manuel Garcia-Ruiz Fermín Otálora Alfonso Garcia CaballeroMass transport takes place within the mesoscopic to macroscopic scale range and plays a key role in crystal growth that may affect the result of the crystallization experiment. The influence of mass transport is different depending on the crystallization technique employed, essentially because each technique reaches supersaturation in its own unique way. In the case of batch experiments, there are some complex phenomena that take place at the interface between solutions upon mixing. These transport instabilities may drastically affect the reproducibility of crystallization experiments, and different outcomes may be obtained depending on whether or not the drop is homogenized. In diffusion experiments with aqueous solutions, evaporation leads to fascinating transport phenomena. When a drop starts to evaporate, there is an increase in concentration near the interface between the drop and the air until a nucleation event eventually takes place. Upon growth, the weight of the floating crystal overcomes the surface tension and the crystal falls to the bottom of the drop. The very growth of the crystal then triggers convective flow and inhomogeneities in supersaturation values in the drop owing to buoyancy of the lighter concentration-depleted solution surrounding the crystal. Finally, the counter-diffusion technique works if, and only if, diffusive mass transport is assured. The technique relies on the propagation of a supersaturation wave that moves across the elongated protein chamber and is the result of the coupling of reaction (crystallization) and diffusion. The goal of this review is to convince protein crystal growers that in spite of the small volume of the typical protein crystallization setup, transport plays a key role in the crystal quality, size and phase in both screening and optimization experiments.ViewShow abstractMicrofluidic Approaches for Protein Crystal Structure AnalysisArticleFull-text availableJan 2016ANAL SCI Masatoshi Maeki Hiroshi Yamaguchi Manabu TokeshiMasaya MiyazakiThis review summarizes two microfluidic-based protein crystallization methods, protein crystallization behavior in the microfluidic devices, and their applications for X-ray crystal structure analysis. Microfluidic devices provide many advantages for protein crystallography; they require small sample volumes, provide high-throughput screening, and allow control of the protein crystallization. A droplet-based protein crystallization method is a useful technique for high-throughput screening and the formation of a single crystal without any complicated device fabrication process. Well-based microfluidic platforms also enable effective protein crystallization. This review also summarizes the protein crystal growth behavior in microfluidic devices as, is known from viewpoints of theoretical and experimental approaches. Finally, we introduce applications of microfluidic devices for on-chip crystal structure analysis.ViewShow abstractCrystal growth under external electric fieldsConference PaperOct 2014 Satoshi UdaHaruhiko Koizumi J. Nozawa Kozo FujiwaraThis is a review article concerning the crystal growth under external electric fields that has been studied in our lab for the past 10 years. An external field is applied electrostaticallyeither through an electrically insulating phase or a direct injection of an electric current to the solid-interface-liquid. The former changes the chemical potential of both solid and liquid and controls the phase relationship while the latter modifies the transport and partitioning of ionic solutes in the oxide melt during crystallization and changes the solute distribution in the crystal.ViewShow abstractGlucose Isomerase Polymorphs Obtained Using an Ad Hoc Protein Crystallization Temperature Device and a Growth Cell Applying an Electric FieldArticleJan 2016Cryst Growth Des Siseth Martínez Caballero Mayra Cuéllar CruzNicola Demitri Abel MorenoViewNon-equilibrium evaporation and condensation at microgravityArticleNov 1995 Sung P. LinM. HudmanAn exact solution of a differential system for a problem of non-equilibrium evaporation or condensation under weightless conditions across an interface between a viscous liquid and a viscous gas is obtained. Numerical results are obtained to delineate the functional relations between relevant parameters and the evaporation coefficient which appears in the theory of non-equilibrium evaporation. Based on the results of the exact solution a conceptual experiment is suggested for determining the evaporation coefficient under weightless conditions. The coefficient determined in a microgravity environment will be free from being masked by the natural convection effect on earth.ViewShow abstractChipping in to microfluidicsArticleSep 2007PHYS WORLDCarl HansenKaston Leung Payam MousaviImagine stepping off the edge of a swimming pool, only to find that your foot deflects the surface of the water without breaking it, as if held by some impenetrable skin. As you walk forward, the water continues to support you; but if you take a running leap and bring your full weight down on the surface, then it snaps open to envelop you without a splash. Rather than plunging to the bottom of the pool, however, you stop abruptly as your kinetic energy is instantaneously dissipated in the fluid. You flail your arms in an attempt to return to the pool s edge but make no progress, merely bouncing back and forth with each stroke.ViewShow abstractBehavior of fluids under high magnetic fieldsArticleJan 1997N.I. WakayamaViewCurrent trends in protein crystallizationArticleDec 2015Arch Biochem Biophys Jose A. GaviraUnlabelled: Proteins belong to the most complex colloidal system in terms of their physicochemical properties, size and conformational-flexibility. This complexity contributes to their great sensitivity to any external change and dictate the uncertainty of crystallization. The need of 3D models to understand their functionality and interaction mechanisms with other neighbouring (macro)molecules has driven the tremendous effort put into the field of crystallography that has also permeated other fields trying to shed some light into reluctant-to-crystallize proteins. This review is aimed at revising protein crystallization from a regular-laboratory point of view. It is also devoted to highlight the latest developments and achievements to produce, identify and deliver high-quality protein crystals for XFEL, Micro-ED or neutron diffraction. The low likelihood of protein crystallization is rationalized by considering the intrinsic polypeptide nature (folded state, surface charge, etc) followed by a description of the standard crystallization methods (batch, vapour diffusion and counter-diffusion), including high throughput advances. Other methodologies aimed at determining protein features in solution (NMR, SAS, DLS) or to gather structural information from single particles such as Cryo-EM are also discussed. Finally, current approaches showing the convergence of different structural biology techniques and the cross-methodologies adaptation to tackle the most difficult problems, are presented.Synopsis: Current advances in biomacromolecules crystallization, from nano crystals for XFEL and Micro-ED to large crystals for neutron diffraction, are covered with special emphasis in methodologies applicable at laboratory scale.ViewShow abstractShow moreAdvertisementRecommendationsDiscover moreProjectEFFECTS OF ELECTROMAGNETIC FIELDS ON PROTEIN CRYSTALLIZATION AND 3D STRUCTURES OF PROTEINS Abel Moreno Juan Pablo Reyes-Grajeda Margarita Rivera[...]David Jáuregui-ZúñigaWe are dealing with the application of strong magnetic fields higher than 15 Tesla in order to control de size and crystal orientation. Additionally, we are applying AC/DC electric fields in order to control the crystal nucleation and growth of model proteins, membrane proteins and macromolecular complexes. ... [more]View projectProject141/5000 Functional and structural studies of the biological macromolecules involved in endothelial dysfunction in cardiovascular pathologies. Azucena Eunice Jimenez Corona Abel Moreno Jaime Mas-Oliva[...]Salvador DamianView projectProjectCrystallogenesis Claude Sauter Bernard Lorber Richard Giegé[...] Juan Manuel Garcia-RuizView projectProjectCandida Mayra Cuéllar Cruz Abel Moreno Nicola Demitri[...] Arturo Vega-GonzalezView projectArticleMössPAC: A UHV-system for surface and thin film investigations using nuclear probesAugust 1995 · VacuumM. SemplePan Min B. Hjörvarsson[...] Erik B KarlssonA UHV-system constructed for surface, thin film and superlattice measurements, using the time-differential perturbed gamma-gamma angular correlations (DPAC) and Mossbauer spectroscopy techniques, is described. Sample preparation is discussed, particularly with regard to the production of suitable radioactive probes. Measurements are described in order to demonstrate the ability to determine local ... [Show full abstract] magnetic fields and electric field gradients. Tests of the system performed on a Cu(111) single crystal reproduced previous results measured by Klas et al. The local magnetic field in thin fee cobalt films on Cu(100) was measured to be 23.0 T, a reduction compared with the hcp bulk Co value. In addition a weak electric field gradient was found, resulting from a tetragonal distortion in the film perpendicular to the surface.Read moreArticleFull-text availableCoupling of a biquaternionic Dirac field to a bosonic fieldAugust 2017 · Theoretical and Mathematical Physics Arbab Ibrahim ArbabWe extend the biquaternionic Dirac equation to include interactions with a background bosonic field. The obtained biquaternionic Dirac equation yields Maxwell-like equations that hold for both a matter field and an electromagnetic field. We establish that the electric field is perpendicular to the matter magnetic field and the magnetic field is perpendicular to the matter inertial field. We show ... [Show full abstract] that the inertial and magnetic masses are conserved separately. The magnetic mass density arises as a result of the coupling between the vector potential and the matter inertial field. The presence of the vector and scalar potentials and also the matter inertial and magnetic fields modify the standard form of the derived Maxwell equations. The resulting interacting electrodynamics equations are generalizations of the equations of Wilczek or Chert–Simons axion-like fields. The coupled field in the biquaternioic Dirac field reconstructs the Wilczek axion field. We show that the electromagnetic field vector F⃗ =E⃗ +icB⃗ , where E⃗ and B⃗ are the respective electric and magnetic fields, satisfies the massive Dirac equation and, moreover, ∇⃗ ⋅F⃗ =0.View full-textArticleEffects of physical environments on nucleation of protein crystals: a reviewJanuary 2011 · Sheng wu gong cheng xue bao = Chinese journal of biotechnology Chen RuiqingJun LiuQinqin Lu[...] Da-Chuan YinThis paper reviews the effects of physical environments (including light, electric field, ultrasound, magnetic field, microgravity, temperature, mechanical vibration, and heterogeneous nucleation interface) on protein crystal nucleation. The research results are summarized and the possible mechanisms of the effects are discussed. In the end of this review, the application prospects of these ... [Show full abstract] physical environments (including coupled environments) in protein crystallization are presented.Read moreArticleNonlinear stress-strain behavior of nematic elastomers using relative rotationsAndreas M MenzelHarald PleinerHelmut R. BrandAs a recent development in the area of side-chain liquid single crystal elastomers, the mate- rials are exposed to an external field of large amplitude to drive them into the nonlinear regime. Then, a second external field is superimposed. The small-amplitude response to this second external field is recorded under the influence of the first, large-amplitude external field. Current experimental ... [Show full abstract] work includes small-amplitude shear measurements (1) as well as dynamic light scattering measurements (2) on prestretched nematic SCLSCEs. In the latter case, essentially the response to externally imposed electromagnetic fields is recorded. We investigate the response of prestretched nematic side-chain single crystal elastomers to superimposed external shear, electric, and magnetic fields of small amplitude. The prestretching direction is oriented perpendicular to the initial nematic director orientation, which enforces director reorientation. Furthermore, the shear plane contains the direction of prestretch. In this case, we obtain a strongly decreased effective shear modulus in the vicinity of the onset and the completion of the enforced director rotation. For the same regions, we find that it becomes comparatively easy to reorient the director by external electric and magnetic fields. These results were derived using conventional elasticity theory and its coupling to relative director-network rotations (3,4). Our results are interesting from an experimental and an applied point of view. Since director reorientations in nematic elastomers are connected to elastic deformations, they are candidates for the potential use as actuators (5-7). So far, only swollen nematic elastomers show a consid- erable director reorientation at reasonably low external electric field amplitudes (6,7). However, fine-tuned prestretching of nematic elastomers may allow a considerable electric field induced director reorientation and resulting elastic deformations also for dry materials.Read moreDiscover the world s researchJoin ResearchGate to find the people and research you need to help your work.Join for free ResearchGate iOS AppGet it from the App Store now.InstallKeep up with your stats and moreAccess scientific knowledge from anywhere orDiscover by subject areaRecruit researchersJoin for freeLoginEmail Tip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? Keep me logged inLog inorContinue with GoogleWelcome back! 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