sign in sign up
<<
Home , Crystallization, Dialysis (biochemistry), Filtration, Supersaturation

Review Choosing the Method of to Obtain Optimal Results

Lata Govada and Naomi E. Chayen * Affiliation Computational and Systems Medicine, Department of Surgery and Cancer, Faculty of Medicine, Review Imperial College London, London SW7 2AZ, UK; [email protected] * Correspondence:Choosing [email protected] the Method of Crystallization to Obtain Optimal Results  Received: 21 January 2019; Accepted: 16 February 2019; Published: 19 February 2019 

Abstract:LataAnyone Govada whoand Naomi has ever E Chayen attempted * to crystallise a or other biological macromolecule has encounteredAffiliation Computational at least one, and if notSystems all Medicine, of the following Department scenarios:of Surgery and No Cancer, crystals Faculty at of all, Medicine, tiny low quality Imperial College London, London SW7 2AZ, UK; [email protected] crystals;* Correspondence: separation; [email protected] amorphous precipitate and the most frustrating; large, beautiful crystals that do not diffract at all. In this paper we review a number of simple ways to overcome such Received: 21 January 2019; Accepted: 16 February 2019; Published: 19 February 2019 problems, which have worked in our hands and in other laboratories. It brings together informationAbstract: that Anyone has been who dispersed has ever attempted in various to crystallise publications a protein and or lecturesother biological over themacromolecule years and includes further informationhas encountered that at hasleast notone, beenif not all previously of the followin published.g scenarios: No crystals at all, tiny low quality crystals; phase separation; amorphous precipitate and the most frustrating; large, beautiful crystals that do not diffract at all. In this paper we review a number of simple ways to overcome such Keywords: problems,; which macromolecules; have worked well crystals; in our hands crystallization; and in other methodology; laboratories. It brings phase together diagram information that has been dispersed in various publications and lectures over the years and includes further information that has not been previously published.

1. IntroductionKeywords: proteins; macromolecules; crystals; crystallization; methodology;

The crystallization phase diagram forms the basis for growth and it is central to every crystallization experiment. Obtaining a phase diagram is commonly done after finding a hit during 1. Introduction the screening experiments by dispensing (manually or by a robot) 10–24 trials varying the protein and precipitant concentrationsThe crystallization in phase steps. diagram Alternatively, forms the the basis pH, for , orand another it is central parameter to every to which crystallization experiment. Obtaining a phase diagram is commonly done after finding a hit during the solubilityscreening is sensitive experiments can be by varied. dispensing These (manually experiments or by a robot) provide 10–24 thetrials super- varying the protein curve, and which separatesprecipitant the nucleation conditions in steps. from Alternatively, the metastable the pH, temperature, conditions or which another are parameter ideal for to which growth [1]. Crystallizationsolubility is sensitive methods can followbe varied. different These experiments routes on provide the phase the diagram.super-solubility In thecurve, case which of the batch method,separates the drops thereach nucleation super-saturation conditions from the instantaneously metastable conditions upon which mixing are ideal the for protein growth [1]. and crystallizing . OnCrystallization the other hand, methods in vapour follow different , routes both on freethe phase interface diagram. diffusion In the case and of the batch trials, the method, the drops reach super-saturation instantaneously upon mixing the protein and crystallizing protein solutionsolutions. is On under-saturated the other hand, in tovapour start diffusion, with, and both attains free interface super-saturation diffusion and on dialysis equilibrating trials, the with the crystallizingprotein solutions overis under-saturated the course ofto start the trialwith, (Figureand attains1). super-saturation on equilibrating with the crystallizing solutions over the course of the trial (Figure 1).

Figure 1.FigurePhase 1.Diagram Phase Diagram illustrating illustrating the the different different routes of of attaining attaining . supersaturation. (i) Micro-batch (i) Micro-batch trials wheretrials instant where instant supersaturation supersaturation is is reached. reached. (ii) Vapour Vapour diffusion diffusion (iii) Dialysis (iii) Dialysis (iv) free (iv) interface free interface diffusiondiffusion trials. trials. The super-solubilityThe super-solubility curve curve separatesseparates the the conditions conditions under under which whichspontaneous spontaneous nucleationnucleation occurs andoccurs the and metastable the metastable zone, zone, ideal ideal for fo crystalsr crystals growth growth [[2].2]. Reprinted Reprinted by by permission permission from Springer [Nature Methods] (Protein crystallization: from purified protein to diffraction-quality Crystals 2019, 9, x; doi: FOR PEER REVIEW www.mdpi.com/journal/crystals crystal, Naomi E Chayen, Emmanuel Saridakis) (2008).

Crystals 2019, 9, 106; doi:10.3390/cryst9020106 www.mdpi.com/journal/crystals Crystals 2019, 9, 106 2 of 7

There are various outcomes when setting up crystallization trials:

• Obtaining no crystals at all. • Obtaining showers of small crystals which are not suitable for diffraction. • Obtaining large crystals or twinned crystals that do not diffract. • Obtaining reasonable crystals that require improvement. • Obtaining good crystals that are unreproducible or difficult to repeat

It is well known that , and its quality, have a crucial impact on the crystallization results. Hence, prior to setting up crystallization trials, it is essential for the protein sample to be as pure as possible (e.g., [3]), to be soluble, and to be stable ([4–6] and references within). A useful approach for establishing the optimal buffer for protein stability is to test a variety of buffer conditions using a thermo-stability assay [7]. Choosing which crystallization method to use depends on the problem, and in this paper, we review a number of simple ways to overcome such problems.

2. Obtaining No Crystals or Crystals that Do Not Diffract in the Screening Stage of Crystallization

2.1. Adopting A Variety of Crystallization Methods The different crystallization methods lead to the formation of crystals via different routes, as described in Section1 above. Vapour diffusion and micro-batch are the most common methods. In the case of a micro-batch, supersaturation is achieved upon mixing, while vapour diffusion is a dynamic system in which a reservoir acts to gradually concentrate the crystallization drop, thus the change in may lead to crystallization. Baldock et al. reported a comparison of screening, using vapour diffusion and variations of the microbatch method thereby concluding that both methods should be applied to ensure all possibilities are covered [8]. Since that study, a variety of new methods for screening have been established, the most common being microfluidics and chips, which in some laboratories are preferred to microbatch, and even vapour diffusion. The first microfluidic device was developed by Hansen et al. for high-throughput screening of protein crystallization. It applied a miniaturized free interface diffusion technique [9]. Following that, more advanced microfluidic systems have been developed [10–12].

2.2. Concentration of Drops that Would Remain Clear Indefinitely Drops, set up for screening experiments, that remain clear after a two-week incubation period, are mostly ignored and considered a ‘dead end’. This is because the protein solution is either under-saturated or in a metastable condition. These clear drops can be utilized in a hanging drop vapour diffusion set up using Easy Xtal crystallization plates. The screw caps, which seal the well of an Easy Xtal tray, can be unsealed by twisting them for a desired period and resealing them again. An under-saturated/metastable trial can thus be driven to supersaturation, resulting in nucleation and subsequent crystal growth Khurshid et al reported 11 new hits on 3 proteins tested [13]. This technique can also be used for optimisation as reported by Govada and Chayen, who obtained better diffracting crystals of the cardiac Myosin Binding Protein-C with a resolution limit of up to 1.5 Å compared to 3.2 Å using conventional techniques, which led to the determination of its structure [14].

3. Obtaining Showers of Small Crystals It is often the case that a large number of very small useless crystals appear in the trial. This can occur because the crystallization process is taking place too rapidly [15] or the crystallization solution contains dust and other random particles that serve as unintended nucleation sites. There is a variety of ways to improve the quality of such crystals. Crystals 2019, 9, 106 3 of 7 Crystals 2019, 9, x FOR PEER REVIEW 3 of 7

3.1. Rigorous It is standard practicepractice toto filter filter the the sample sample solution solution though though a 0.2a 0.2µm µm mesh mesh size size filter filter before before setting setting up upthe the crystallization crystallization trials, trials, but often but often this is this not sufficientis not sufficient and filtration and filtration needs to needs be done to more be done rigorously. more rigorously.Filtration is Filtration relevant tois allrelevant methods to all of methods crystallization of crystallization at the optimization at the optimization stage. Filtration stage. of Filtration a protein ofsample a protein with sample or without with its or crystallizing without its agents,crystallizing through agents, filters through of different filters sizes of (e.g.,different from sizes 0.1 µ (e.g.,m to 100KDafrom 0.1 filters), µm to can100KDa determine filters), the can number, determine the size th ande number, quality the of the size resulting and qu crystals.ality of the The resulting filtration crystals.can remove The unwantedfiltration can material remove and unwanted particles material such as and dust, particles protein such aggregates as dust, protein and fungi aggregates thereby andreducing fungi the thereby numbers reducing of crystals the numbers from many of crystals useless onesfrom tomany a few useless single ones diffracting to a few ones single and diffracting increasing onesthe reproducibility and increasing of the the reproducibility experiments. Filtration, of the expe doneriments. prior Filtration, to setting updone the prior experiments, to setting does up notthe experiments,necessitate any does change not tonecessitat the crystallizatione any change method, to the vesselcrystallizat or theion conditions. method, Itthe can, vessel therefore, or the be conditions.used as a one-step It can, optimizationtherefore, be strategy used as when a one- numerousstep optimization small worthless strategy crystals, when twinned numerous crystals, small or worthlesseven precipitates crystals, are twinned formed crystals, and no or improvement even precipitates is obtained are formed by conventional and no improvement means. For is obtained example, byfine-tuning conventional the concentrations means. For example, of the protein fine-tuning and crystallizing the concentrations agents, inserting of the protein additives, and changingcrystallizing pH agents,or temperature. inserting Rigorousadditives, filtration changing is pH also or very temperature. useful after Rigorous the storage filtration of proteins is also very and isuseful especially after thevaluable storage in improvingof proteins the and results is especially of seeding valuab and theleapplication in improving of nucleants the results [16 ].of seeding and the application of nucleants [16]. 3.2. Slowing Down the Crystallization Process 3.2. Slowing Down the Crystallization Process A means to improving the size and quality of crystals, which appear as showers of small crystals, twinnedA means crystals to improving or even a crystalline the size and precipitate, quality of iscrys donetals, by which inserting appear an oilas showers barrier over of small the reservoircrystals, twinnedof vapour crystals diffusion or even hanging a crystalline drops or precipitate, sitting drops is done trials. by The inserting barrier an affects oil barrier the rate over of equilibration.the reservoir ofThe vapour speed diffusion of equilibration hanging depends drops onor sitting the ratio drops of paraffin trials. The to silicon barrier oils affects and onthe the rate thickness of equilibration. of the oil Thelayer. speed Using of Linbroequilibration or the Qiagendepends Easy on the X-tal ratio plates of para a layerffin ofto 250siliconµL ofoils a and on the of 1:1thickness paraffin of andthe oilsilicon layer. oils Using works Linbro well inor most the Qiagen cases. TheEasy trials X-tal are plates set up a layer in the of same 250 conditionsµL of a mixture in the of control 1:1 paraffin and in andtrials silicon containing oils works the oil well barrier, in most the only cases. difference The trials is the are presence set up in of the the same layer ofconditions oil above in the the reservoirs. control andThe limitationin trials containing of this technique the oil barrier, is that itthe cannot only diffe be usedrence with is the PEG presence or MPD of concentrations the layer of oil above above 13%, the reservoirs.but it is very The effective limitation at concentrations of this technique below is that 13%, it cannot and at be all used concentrations with PEG ofor allMPD salts concentrations [17]. In trials abovecontaining 13%, an but oil it barrier, is very crystalseffective require at concentrations longer periods below (e.g., 13%, 8–10 and days at comparedall concentrations to 12–24 of h) all to growsalts [17].to full In size, trials but containing their quality an isoil more barrier, improved. crystals One require example longer are periods the crystals (e.g., of 8–10 lobster days apocrustacyanin compared to 12–24C1 that hours) are grown to grow in to trials full withsize, anbut oil their barrier, qualit whichy is more diffract improved. to higher One resolution, example are compared the crystals to the of lobstercontrols apocrustacyanin (1.3 Å compared C1 to 2.1that Å) are grown in trials with an oil barrier, which diffract to higher resolution,Further compared examples to wherethe controls placing (1.3Å an compared oil barrier to over 2.1Å) the reservoir dramatically reduced the nucleation,Further were examples the rat where liver ribosomalplacing an P2 oil protein barrier thereby over the resulting reservoir in higherdramatically diffracting reduced crystals, the nucleation,with a resolution were limitthe rat of liver 2.4 Å ribosomal (Figure2)[ P218 ]protein and GH42 thereby intracellular resulting beta-galactosidase in higher diffracting diffracting crystals, to with2.45 Åa [resolution19]. limit of 2.4 Å (Figure 2) [18] and GH42 intracellular beta-galactosidase diffracting to 2.45Å [19].

(a) (b)

Figure 2. CrystalsCrystals of of an an N-terminal N-terminal fragment fragment of rat liv liverer ribosomal P2 protein with and without an oil barrier. ( a) Control (b) with oil barrier [[18].18].

At the moment, such trials can only be done ma manually,nually, but an oil barrier technique, which is miniaturized and automated, will soon be published.

3.3. Separation of the Nucleation and Growth Stages of Crystallization

Crystals 2019, 9, 106 4 of 7

Crystals 2019, 9, x FOR PEER REVIEW 4 of 7 3.3. Separation of the Nucleation and Growth Stages of Crystallization Volume Adjustments during Crystallization Volume Adjustments during Crystallization It is well documented that nucleation requires different condition to those of growth, therefore methodsIt is wellthat separate documented the phases that nucleation of nucleation requires and growth different can condition lead to remarkable to those of growth,results. therefore methodsOne thatway separate of achieving the phases such separation, of nucleation is andby diluti growthon canof trials, lead toin remarkableorder to transfer results. the system fromOne nucleation way of to achieving metastable such conditions separation, before is by dilutionnucleation of trials,becomes in order excessive. to transfer This is the an system alternative from meansnucleation of achieving to metastable a similar conditions outcome before to seeding, nucleation but wi becomesthout the excessive. need to handle This is crystals an alternative or nucleants. means Whenof achieving the clusters a similar of non-diffracting outcome to seeding, crystals, but whic withouth cannot the need be improved to handle by crystals conventional or nucleants. fine-tuning When ofthe the clusters crystallisation of non-diffracting parameters crystals, are produced, which cannot a working be improved phase bydiagram conventional that provides fine-tuning the super- of the solubilitycrystallisation curve parameters can be generated are produced, (either manually a working or phase using diagram a robot) that (Figure provides 1). the super-solubility curveThe can experiments be generated related (either manuallyto microbatch or using were a robot)reported (Figure by 1Saridakis). et al back in 1994. The AuthorsThe have experiments established related a working to microbatch phase diagram were reported for the enzyme by Saridakis carboxypeptidase et al backin G2, 1994. with ThepH Authorsand temperature have established being kept a constant, working phasewhile varying diagram th fore concentrations the enzyme carboxypeptidase of the protein and G2, precipitant. with pH Theand temperatureworking phase being diagram kept constant, produced while the super-solubility varying the concentrations curve and ofestablished the protein the and conditions precipitant. of Thenucleation working (conditions phase diagram that would produced produce the crystals super-solubility spontaneously). curve and Crystallization established trials the conditions were set up of atnucleation these conditions, (conditions using that a wouldcrystallization produce robot crystals and spontaneously). at various time Crystallization intervals after trialsthe set-up were setof the up experiments,at these conditions, the robot using was a programmed crystallization to robot add a and buffe atr various or protein time solution intervals resulting after the in set-up the dilution of the ofexperiments, the trials. Single the robot diffracting was programmed crystals were to add routinely a buffer orattained, protein equivalent solution resulting to the best, in the very dilution rarely of withoutthe trials. using Single the diffracting dilution procedure crystals were [20,21]. routinely attained, equivalent to the best, very rarely without usingIn the the dilution case of procedure vapour diffusion, [20,21]. the crystallisation drops were not physically diluted, but coverslipsIn the holding case of vapour hanging diffusion, drops thewere crystallisation incubated over drops reservoir were not solutions physically that diluted, would but give coverslips many smallholding crystals. hanging At drops time were intervals incubated (before over crystals reservoir are solutions visible), that the would coverslips give many are smalltransferred crystals. over At reservoirs,time intervals containing (before crystals lower areprecipitant visible), theconcentrat coverslipsions are that transferred would normally over reservoirs, yield clear containing drops lower [22]. precipitantThis technique concentrations has produced that significant would normally improvement yield clear in crystal drops order [22]. Thisof a techniquenumber of hasproteins produced e.g., *MtCMsignificant (Figure improvement 3) [23] inand crystal most order remarkably, of a number crystals of proteins of C-phycocyanin, e.g., *MtCM (Figure which3) [reproducibly23] and most diffractedremarkably, to crystals 1.45 Å, ofcompared C-phycocyanin, to 2 Å, whichusing reproduciblystandard optimisation diffracted totechniques 1.45 Å, compared [24]. This to resolution 2 Å, using wasstandard the highest optimisation ever obtained techniques for [this24]. protein. This resolution was the highest ever obtained for this protein.

Figure 3. *MtCM*MtCM crystals crystals obtained obtained by by transfer transfer from from higher higher to lower to lower precipitant precipitant concentration. concentration. (a) (Controla) Control (b) (afterb) after transfer transfer [23]. [23 ].

4. The Application of Light-Scattering Techniques

Crystals 2019, 9, 106 5 of 7

4. The Application of Light-Scattering Techniques The short-comings of the volume adjustment and transfer techniques, using any crystallization method, is that one does not know when nucleation takes place. It can only be estimated by reference to the time that it took to see the first crystals. The most effective moment to intervene with a crystallisation experiment is soon after the formation of the first critical size nuclei, which will eventually form the crystal. By the time crystals are observed under the , it is too late to act, as nuclei and crystals have already been formed [25]. A means to pinpoint the appropriate time for transfer/dilution or arrest of nucleation, is by using dynamic light scattering (DLS) and static light scattering. DLS offers a size resolution of particles in optically transparent aqueous samples, some three orders of magnitude below an optical microscope. It is therefore a useful tool for an early, non-invasive, in situ observation of a crystallisation event, before it becomes visible with a light microscope [21,26–28]. Light scattering was applied to separate nucleation and growth in the crystallization of lysozyme by Rosenberger et al. who successfully limited the number of crystals that were grown [29]. The procedure involved scintillation, and required 50–100 µL of a sample, which was acceptable at the time, but the procedure needed to be improved and miniaturised for application to other proteins. Almost 10 years later, Saridakis et al. used DLS to monitor the crystallisation of proteins that were mixed with their crystallising agents in microbatch, with the aim of getting an indication as to when to dilute the trial in order to lead it out of the nucleation phase and into the growth phase. This was achieved using a DLS-apparatus (Made by RiNA GmbH, Berlin, Germany), which is able to measure a crystallisation trial as it takes place in standard cuvettes containing 10–20 µL. When the DLS spectrum showed a change in the size-distribution profile of species in the solution, the trial was diluted with buffer to metastable conditions, leading to the growth of fewer and larger crystals, compared to crystals grown in control solutions that were left undisturbed [Patent of Chayen, Dieckmann and Dierks [30]. In the case of vapour diffusion, Collingthworth et al. applied static light scattering to detect a change in the profile of crystallising solutions. The drops were evaporated using nitrogen flow and the was arrested when the light-scattering sensor detected aggregation [31]. Varying the evaporation rate of the crystallisation solutions resulted in the formation of fewer, larger crystals than those obtained in controls. Both dynamic and static light-scattering techniques have more recently been adapted for automated routine use in a micro-batch, vapour diffusion, and other crystallisation set-ups. Transfer from nucleation to metastable conditions can now be achieved in nanoscale and picolitre volumes, by changing parameters such as pH, temperature, humidity, and other parameters, as well as the protein and precipitant concentrations [32]. Recent advances in DLS are also able to provide new and detailed insights of two-step crystal nucleation mechanisms [33].

5. Summary This article highlights the progress that has gradually been made in the design of methods, by describing a selection of the original methods some, which have led to practices that are much improved technically, but are based on the same principles and ideas. The techniques have been miniaturised and automated to nanoscale by using robotics, special crystallisation trays, microfluidics, and micro-chips, thereby also increasing the reproducibility of the results. Moreover, combinations of techniques, such as vapour batch, gels and oils [34] as well as conbining counter-diffusion and microseeding [35,36] are increasingly being used. Other emerging techniques involve crystallization in electric fields [37], as well as preparing crystals for X-ray Free Electron Laser (XFEL), which are outside the scope of this review [38,39].

Funding: The UK and Physical Sciences Research Council. Acknowledgments: We are grateful to the UK Engineering and Physical Sciences Research Council (EPSRC) for financial support. Crystals 2019, 9, 106 6 of 7

Conflicts of Interest: The authors declare no conflict of interest.

References

1. Saridakis, E.; Chayen, N.E. Systematic improvement of protein crystals by determining the supersolubility curves of phase diagrams. Biophys. J. 2003, 84, 1218–1222. [CrossRef] 2. Chayen, N.E.; Saridakis, E. Protein crystallization: From purified protein to diffraction-quality crystal. Nat. Methods 2008, 5, 147–153. [CrossRef][PubMed] 3. Giegé, R.; Dock, A.C.; Kern, D.; Lorber, B.; Thierry, J.C.; Moras, D. The role of purification in the crystallization of proteins and nucleic acids. J. Cryst. Growth 1986, 76, 554–561. [CrossRef] 4. Bergfors, T.M. Protein Crystallization. Techniques, Strategies, and Tips; Bergfors, T.M., Ed.; International University Line: San Diego, CA, USA, 1999. 5. McPherson, A. Crystallization of Biological Macromolecules; Cold Spring Harbor Lab Press: Cold Spring Harbor, NY, USA, 1999. 6. Ducruix, A.; Giegé, R. Crystallization of Nucleic Acids and Proteins: A Practical Approach, 2nd ed.; Oxford University Press: Oxford, UK, 1999. 7. Ericsson, U.B.; Hallberg, B.M.; DeTitta, G.T.; Dekker, N.; Nordlund, P. Thermofluor-based high-throughput stability optimization of proteins for structural studies. Anal. Biochem. 2006, 357, 289–298. [CrossRef] [PubMed] 8. Baldock, P.; Mills, V.; Stewart, P.S. A comparison of microbatch and vapour diffusion for initial screening of crystallization conditions. J. Cryst. Growth 1996, 168, 170–174. [CrossRef] 9. Hansen, C.L.; Skordalakes, E.; Berger, J.M.; Quake, S.R. A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion. Proc. Natl. Acad. Sci. USA 2002, 99, 16531. [CrossRef][PubMed] 10. Sauter, C.; Dhouib, K.; Lorber, B. From macrofluidics to microfluidics for the crystallization of biological macromolecules. Cryst. Growth Des. 2007, 7, 2247–2250. [CrossRef] 11. Abdallah, B.G.; Roy-Chowdhury, S.; Fromme, R.; Fromme, P.; Ros, A. Protein Crystallization in an Actuated Microfluidic Nanowell Device. Cryst. Growth Des. 2016, 16, 2074–2082. [CrossRef] 12. Otálora, F.; Gavira, J.A.; Ng, J.D.; García-Ruiz, J.M. Counterdiffusion methods applied to protein crystallization. Prog. Biophys. Mol. Biol. 2009, 101, 26–37. [CrossRef] 13. Khurshid, S.; Govada, L.; Chayen, N.E. Dynamic Screening Experiments to Maximize Hits for Crystallization. Cryst. Growth Des. 2007, 7, 2171–2175. [CrossRef] 14. Govada, L.; Chayen, N.E. Crystallization by Controlled Evaporation Leading to High Resolution Crystals of the C1 Domain of Cardiac Myosin Binding Protein-C (cMyBP-C). Cryst. Growth Des. 2009, 9, 1729–1732. [CrossRef] 15. Chayen, N.E. The role of oil in macromolecular crystallization. Structure 1997, 5, 1269–1274. [CrossRef] 16. Chayen, N.E. Rigorous filtration for protein crystallization. J. Appl. Crystallogr. 2009, 42, 743–744. [CrossRef] 17. Chayen, N.E. A novel technique to control the rate of vapour diffusion, giving larger protein crystals. J. Appl. Crystallogr. 1997, 30, 198–202. [CrossRef] 18. Mandelman, D.; Gonzalo, P.; Lavergne, J.P.; Corbier, C.; Reboud, J.P.; Haser, R. Crystallization and preliminary X-ray study of an N-terminal fragment of rat liver ribosomal P2 protein. Acta Crystallogr. D 2002, 58, 668–671. [CrossRef][PubMed] 19. Solomon, H.V.; Tabachnikov, O.; Feinberg, H.; Govada, L.; Chayen, N.E.; Shoham, Y.; Shoham, G. Crystallization and preliminary crystallographic analysis of GanB, a GH42 intracellular beta-galactosidase from Geobacillus stearothermophilus. Acta Crystallogr. F 2013, 69, 1114–1119. [CrossRef] 20. Saridakis, E.E.G.; Stewart, P.D.S.; Lloyd, L.F.; Blow, D.M. Phase-Diagram and Dilution Experiments in the Crystallization of Carboxypeptidase-G(2). Acta Crystallogr. D 1994, 50, 293–297. [CrossRef][PubMed] 21. Chayen, N.E. Methods for separating nucleation and growth in protein crystallisation. Prog. Biophys. Mol. Biol. 2005, 88, 329–337. [CrossRef] 22. Saridakis, E.; Chayen, N.E. Improving protein crystal quality by decoupling nucleation and growth in diffusion. Protein Sci. 2000, 9, 755–757. [CrossRef] Crystals 2019, 9, 106 7 of 7

23. Krengel, U.; Dey, R.; Sasso, S.; Okvist, M.; Ramakrishnan, C.; Kast, P. Preliminary X-ray crystallographic analysis of the secreted chorismate mutase from Mycobacterium tuberculosis: A tricky crystallization problem solved. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2006, 62, 441–445. [CrossRef] 24. Nield, J.; Rizkallah, P.; Barber, J.; Chayen, N.E. The 1.45 Angstrom three-dimensional structure of C-phycocyanin from the thermophilic cyanobacterium Synechococcus elongatus. J. Struct. Biol. 2003, 141, 149–155. [CrossRef] 25. Saridakis, E.; Dierks, K.; Moreno, A.; Dieckmann, M.W.M.; Chayen, N.E. Separating nucleation and growth in protein crystallization using dynamic light scattering. Acta Crystallogr. D 2002, 58, 1597–1600. [CrossRef] [PubMed] 26. Shmitz, S. An introduction to Dynamic Light Scattering by Macromoelcules. Phys. Today 1990, 44, 66. [CrossRef] 27. Dierks, K.; Meyer, A.; Einspahr, H.; Betzel, C. Dynamic Light Scattering in Protein Crystallization Droplets: Adaptations for Analysis and Optimization of Crystallization Processes. Cryst. Growth Des. 2008, 8, 1628–1634. [CrossRef] 28. Proteau, A.; Shi, R.; Cygler, M. Application of Dynamic Light Scattering in Protein Crystallization. Curr. Protoc. Protein Sci. 2010, 61, 10–17. 29. Rosenberger, F.; Howard, S.B.; Sowers, J.W.; Nyce, T.A. Temperature dependence of protein solubility—Determination and application to crystallization in X-ray capillaries. J. Cryst. Growth 1993, 129, 1–12. [CrossRef] 30. Chayen, N.E.; Dieckmann, M.; Dierks, K. Use of Dynamic Light Scattering in a Method for Producing Macromolecular Crystals. WO 03080901 A1, 23 May 2002. 31. Collingsworth, P.; Bray, T.; K Christopher, G. Crystal Growth via Computer Controlled Vapor Diffusion. J. Cryst. Growth 2000, 219, 283–289. [CrossRef] 32. Meyer, A.; Dierks, K.; Hilterhaus, D.; Klupsch, T.; Muhlig, P.; Kleesiek, J.; Schopflin, R.; Einspahr, H.; Hilgenfeld, R.; Betzel, C. Single-drop optimization of protein crystallization. Acta Crystallogr. Sect. F 2012, 68, 994–998. [CrossRef] 33. Schubert, R.; Meyer, A.; Baitan, D.; Dierks, K.; Perbandt, M.; Betzel, C. Real-Time Observation of Protein Dense Cluster Evolution during Nucleation in Protein Crystallization. Cryst. Growth Des. 2017, 17, 954–958. [CrossRef] 34. Moreno, A.; Saridakis, E.; Chayen, N.E. Combination of oils and gels for enhancing the growth of protein crystals. J. Appl. Crystallogr. 2002, 35, 140–142. [CrossRef] 35. Gavira, J.A.; Hernandez-Hernandez, M.A.; Gonzalez-Ramirez, L.A.; Briggs, R.A.; Kolek, S.A.; Shaw Stewart, P.D. Combining Counter-Diffusion and Microseeding to Increase the Success Rate in Protein Crystallization. Cryst. Growth Des. 2011, 11, 2122–2126. [CrossRef] 36. Bolanos-Garcia, V.M.; Chayen, N.E. New directions in conventional methods of protein crystallization. Prog. Biophys. Mol. Biol. 2009, 101, 3–12. [CrossRef][PubMed] 37. Moreno, A. Protein Crystallization under the Presence of an Electric Field; MDPI Books: Basel, Switzerland, 2018. 38. Johansson, L.C.; Stauch, B.; Ishchenko, A.; Cherezov, V. A Bright Future for Serial Femtosecond with XFELs. Trends Biochem. Sci. 2017, 42, 749–762. [CrossRef] 39. McSweeney, S.; Fromme, P. Crystallography: Sources of inspiration. Nature 2014, 505, 620–621. [CrossRef] [PubMed]

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).