MBS acknowledges the support from the LABEX VALO GRAL under the contract 2015. NJ acknowledges CEA&#39;s International Doctoral Research Program (Irtelis) for the PhD Fellowship. Authors acknowledge funding from the European Union&#8217;s Horizon 2020 Research and Innovation Program under Marie Sk&#322;odowska-Curie grant agreement number 722687. Authors are also grateful to Dr Esko Oksanen (ESS, Lund) and Dr Jean-Luc Ferrer (IBS, Grenoble) for helpful conversations and insights. IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA).

<ol>
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A., Huang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-volume+protein+crystal+growth+for+neutron+macromolecular+crystallography.">Large-volume protein crystal growth for neutron macromolecular crystallography.</a> Acta Crystallographica Section F:Structural Biology Communications. 71, 358-370 (2015).</li><li>O'Dell, W. B., Bodenheimer, A. M., Meilleur, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutron+protein+crystallography:+A+complementary+tool+for+locating+hydrogens+in+proteins.">Neutron protein crystallography: A complementary tool for locating hydrogens in proteins.</a> Archives of Biochemistry and Biophysics. 602, 48-60 (2016).</li><li>Oksanen, E., Blakeley, M. P., Bonnet&#233;, F., Dauvergne, M. T., Dauvergne, F., Budayova-Spano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+crystal+growth+by+thermal+control+allows+combined+X-ray+and+neutron+crystallographic+studies+to+elucidate+the+protonation+states+in+Aspergillus+flavus+urate+oxidase.">Large crystal growth by thermal control allows combined X-ray and neutron crystallographic studies to elucidate the protonation states in Aspergillus flavus urate oxidase.</a> Journal of the Royal Society Interface. 6, (2009).</li><li>Krauss, I. R., Merlino, A., Vergara, A., Sica, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+biological+macromolecule+crystallization.">An overview of biological macromolecule crystallization.</a> International Journal of Molecular Sciences. 14 (6), 11643-11691 (2013).</li><li>Chayen, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+Studies+of+Protein+Crystallization+by+Vapour-Diffusion+and+Microbatch+Techniques.">Comparative Studies of Protein Crystallization by Vapour-Diffusion and Microbatch Techniques.</a> REVIEW Acta Cryst. , (1998).</li><li>Junius, N., Oksanen, E., Terrien, M., Berzin, C., Ferrer, J. L., Budayova-Spano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+crystallization+apparatus+for+temperaturecontrolled+flow-cell+dialysis+with+real-time+visualization.">A crystallization apparatus for temperaturecontrolled flow-cell dialysis with real-time visualization.</a> Journal of Applied Crystallography. 49, 806-813 (2016).</li><li>Salemme, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+free+interface+diffusion+technique+for+the+crystallization+of+proteins+for+X-ray+crystallography.">A free interface diffusion technique for the crystallization of proteins for X-ray crystallography.</a> Archives of Biochemistry and Biophysics. 151 (2), 533-539 (1972).</li><li>Chayen, N. E., Saridakis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+crystallization:+From+purified+protein+to+diffraction-quality+crystal.">Protein crystallization: From purified protein to diffraction-quality crystal.</a> Nature Methods. 5 (2), 147-153 (2008).</li><li>Ot&#225;lora, F., Gavira, J. A., Ng, J. D., Garc&#237;a-Ruiz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counterdiffusion+methods+applied+to+protein+crystallization.">Counterdiffusion methods applied to protein crystallization.</a> Progress in Biophysics and Molecular Biology. 101 (1-3), 26-37 (2009).</li><li>Garci&#225;-Ruiz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counterdiffusion+Methods+for+Macromolecular+Crystallization.">Counterdiffusion Methods for Macromolecular Crystallization.</a> Methods in Enzymology. 368, 130-154 (2003).</li><li>Budayova-Spano, M., Koruza, K., Fisher, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+crystal+growth+for+neutron+protein+crystallography.">Large crystal growth for neutron protein crystallography.</a> Methods in Enzymology. 634, 21-46 (2020).</li><li>Budayova-Spano, M., Dauvergne, F., Audiffren, M., Bactivelane, T., Cusack, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+methodology+and+an+instrument+for+the+temperature-controlled+optimization+of+crystal+growth.">A methodology and an instrument for the temperature-controlled optimization of crystal growth.</a> Acta Crystallographica Section D: Biological Crystallography. 63 (3), 339-347 (2007).</li><li>Asherie, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+crystallization+and+phase+diagrams.">Protein crystallization and phase diagrams.</a> Methods. 34 (3), 266-272 (2004).</li><li>Astier, J. P., Veesler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+temperature+to+crystallize+proteins:+A+mini-review.">Using temperature to crystallize proteins: A mini-review.</a> Crystal Growth and Design. 8 (12), 4215-4219 (2008).</li><li>Budayova-Spano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+preliminary+neutron+diffraction+study+of+rasburicase,+a+recombinant+urate+oxidase+enzyme,+complexed+with+8-azaxanthin.">A preliminary neutron diffraction study of rasburicase, a recombinant urate oxidase enzyme, complexed with 8-azaxanthin.</a> Acta Crystallographica Section F: Structural Biology and Crystallization Communications. 62 (3), 306-309 (2006).</li><li>Junius, N., Vahdatahar, E., Oksanen, E., Ferrer, J. L., Budayova-Spano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+crystallization+of+biological+macromolecules+using+dialysis+combined+with+temperature+control.">Optimization of crystallization of biological macromolecules using dialysis combined with temperature control.</a> Journal of Applied Crystallography. 53 (3), (2020).</li><li>Grimsley, G. R., Pace, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+Determination+of+Protein+Concentration.">Spectrophotometric Determination of Protein Concentration.</a> Current Protocols in Protein Science. 33 (1), 1-9 (2003).</li><li>McPherson, A., Gavira, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+protein+crystallization.">Introduction to protein crystallization.</a> Acta Crystallographica Section F:Structural Biology Communications. 70 (1), 2-20 (2014).</li><li>McPherson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+protein+crystallization.">Introduction to protein crystallization.</a> Methods. 34 (3), 254-265 (2004).</li></ol>

The authors have nothing to disclose.

Different physical, chemical and biological variables influence protein crystallization by affecting protein solubility21. Among these variables, temperature and chemical composition of the crystallization solution are used here in combination with dialysis technique to improve and grow large high-quality crystals of biomacromolecules for neutron diffraction studies. By using knowledge of phase diagrams, crystallization is made more predictable. Although screening of different crystallization conditions in a serial approach is also possible, the main aim of using the rational approaches presented is to separate and control the kinetics of crystal nucleation and growth.
Similar to all crystallization studies, high quality pure and homogeneous protein samples, and dust-free crystallization solutions increase the success rate of the experiment. Filtration and centrifugation of solutions are essential steps in the described protocols. Knowing the physicochemical properties of the proteins studied such as the molecular weight (to choose the appropriate dialysis membrane), the isoelectric point, and the protein solubility are crucial for the design of an optimal crystal growth experiment. Also, consideration must be made for protein stability at different temperatures or with different chemicals to prevent sample loss and increase the likelihood of success. Considering the temperature range of OptiCrys (233.0&#8211;353.0 &#177; 0.1 K), a broad range of proteins can be crystallized using it. But it is worth to stress that proteins that are primarily thermo-stable, such as proteins from thermophilic sources, would benefit the most in temperature-controlled large-volume crystal growth experiments offered by this instrument.
Using a low-volume dialysis chamber (when using OptiCrys) or microdialysis buttons and screening several temperatures and crystallization conditions (e.g., grids of precipitant concentration or pH), it is possible to gain information on the location of the limit of the metastable zone (kinetic equilibrium between nucleation and metastable zones). This is invaluable when designing a successful crystal growth experiment especially for new protein candidates in crystallization. Without this information experiments can start from an area of the phase diagram with high supersaturation, too far from the limit of the metastable zone to easily control crystal nucleation. Although dissolution of the protein precipitate may be attempted, for example by increasing the temperature in the case of direct solubility, for proteins with reduced thermostability, keeping the sample at high temperature for a longer period of time may render the protein precipitation irreversible. Thus, the best strategy consists of using an initial condition with lower supersaturation located near the limit of metastability, where nucleation can be controlled and protein precipitation avoided. In line with this, crystallization prescreening decreases the chance of having a protein precipitate in the dialysis chamber and increases the success rate of the experiment.
After designing an experiment, preparing dialysis chambers (OptiCrys) or microdialysis buttons is another important step. Preventing air bubble formation in the dialysis chamber/button increases the chance of successful crystallization especially when small volumes are used. The presence of air bubbles in dialysis chamber may also change the kinetics of the crystallization process and reduce the reproducibility of the experiment (because the protein/solution contact surface has been modified). Not only protein but also crystallization solution can affect the success of the experiment. Using new 50 mL tubes for the pumping system each time one wants to start a new experiment and washing tubing after each experiment decreases the chance of contamination and avoids the creation of salt crystals in the apparatus.
The use of microdialysis buttons is an alternative when OptiCrys is not available. The strategies for optimizing crystallization and monitoring crystal growth mentioned above, must be carried out manually. Typically this necessitates being outside a thermoregulated incubator, which can be problematic when temperature regulation is a critical step in the methodology described. This does not facilitate changing the chemical composition of the crystallization solutions, or monitoring crystal growth by imaging, so the crystal growth process cannot be controlled in real-time.
Knowledge of the phase diagram is the basis of using the crystallization bench, OptiCrys, to systematically grow large, high-quality crystals in an automated fashion. Control of physicochemical parameters like temperature, precipitant concentration, and pH during crystallization moves the protein-solution equilibrium in a well-defined kinetic trajectory across the phase diagram. This is complemented by the use of a dialysis membrane to adjust mass transport and create a controlled gradient in the crystallization chamber that affects the size and quality of the crystals. Therefore, using both thermodynamic data and kinetic trajectories is essential to control the crystallization process in order to grow high-quality crystals. Thanks to OptiCrys, systematic phase diagrams in a multidimensional space can be studied with a serial approach using significantly less material than before. To demonstrate this methodology, we provide here a case study with a model protein, chicken egg-white lysozyme. By using and mastering the protocol presented here one can adapt it for real protein systems5,14,17,18.

Understanding the structure-function relationship of proteins and the mechanism of physiological pathways often relies on knowing the positions of hydrogen atoms (H) and how charge is transferred within a protein1,2. Since hydrogen atoms scatter X-rays weakly, their positions can only be determined with very high-resolution X-ray diffraction data (&#62;1 &#197;)3,4. Conversely, neutron crystallography can be used to obtain an accurate position of hydrogen atoms in biological macromolecules as hydrogen and deuterium (H2, isotope of hydrogen) atoms have scattering lengths of roughly equal magnitude as oxygen, nitrogen and carbon5. However, neutron flux from available neutron sources is weaker than that of X-ray beams, so this must often be compensated for2,3. This can be achieved by exchanging H with H2 and/or increasing the volume of crystals to reduce the incoherent scattering of hydrogens and increase the signal-to-noise ratio of diffraction images.
There are various crystallization approaches (the corresponding schematic phase diagram is shown in Figure 1) for obtaining large and high-quality crystals for both X-ray and neutron bio-macromolecular crystallography6. In vapor diffusion, a droplet prepared from a mixture of a protein and a crystallization solution is equilibrated over time, through evaporation of water or other volatile species, against a reservoir containing a higher concentration of precipitant of the same crystallization solution. The increase in concentration of protein and precipitant in the droplet leads to the supersaturation required for spontaneous nucleation followed by crystal growth at these nuclei6,7. Although vapor diffusion is the most frequently used technique for growing crystals4, the crystallization process cannot be precisely controlled8. In the free interface diffusion method, crystallization solution diffuses into a concentrated protein solution, very slowly directing the system towards supersaturation. This method can be considered as a batch method with a slow mixing rate6,9,10,11,12. In the batch method, the protein is rapidly mixed with a crystallization solution leading to rapid supersaturation and in turn uniform nucleation with many crystals3,7. This method accounts for approximately one-third of all structures currently deposited in the Protein Data Bank. The dialysis method is also used for growing high-quality and well-diffracting protein crystals. In the dialysis method, molecules of precipitant diffuse from a reservoir through a semi-permeable membrane into a separate chamber with the protein solution. The kinetics of equilibration is dependent on various factors, such as temperature, membrane pore size, and the volume and concentration of protein samples and crystallization agents6.
Crystallization phase diagrams can be used to describe different states of a protein as a function of different physical or chemical variable3. As illustrated in Figure 1, each crystallization technique can be visualized as using a different kinetic trajectory to reach the nucleation and metastable zones of such a diagram6,10,13. This provides information about protein solubility and the protein concentration at which a thermodynamic equilibrium between crystal and solution is observed, thereby finding the optimal conditions for nucleation and growth3,14. In a two-dimensional phase diagram, the protein concentration is plotted as a function of one variable and the other variables are kept constant15. In such a phase diagram, when the protein concentration is below the solubility curve, the solution is in the undersaturated region and no nucleation&#160;or crystal growth occurs. Above this curve is the supersaturation zone where the protein concentration is higher than the solubility limit3,14. This is further divided into three regions: the metastable zone, the spontaneous nucleation zone, and the precipitation zone. In the metastable zone, supersaturation is not sufficient for nucleation to occur within a reasonable time but growth of seeded crystals can take place. Aggregation and precipitation are favored in the precipitation zone, where supersaturation is too high14,15.
When sufficient supersaturation for spontaneous nucleation is achieved, the first nuclei will appear10. The growth of crystals leads to a reduction in the protein concentration until the limit of solubility is reached. As long as supersaturation stays in the vicinity of the solubility curve, there will be no significant change in the size of crystals. However, it has been shown that variations in the temperature and chemical composition of crystallization solution (for example, the concentration of precipitant) will affect protein solubility and may lead to the initiation of further crystal growth8,13,16.
As dialysis is advantageous for good quality crystal growth, the OptiCrys crystallization bench illustrated in Figure 2, was designed and developed in our laboratory to control crystallization in a fully automated manner8. For this purpose, software was written with LabVIEW that allows the control and monitoring of the temperature of a flowing reservoir dialysis setup in contact with Peltier elements, via an electronic controller and a chiller. The same software also automatically regulates the chemical composition of the crystallization solution (for example the exchange of crystallization agents) using a multichannel fluidic system. Additionally, a digital camera and an inverted microscope are used to visualize and record the crystallization process. Two crystallization chambers with 15 &#956;L and 250 &#956;L volumes are available for growing crystals for different purposes. As the crystallization process is reversible, screening for different conditions is possible with just a few microliters of the protein solution as long as the sample is not damaged8. As a result, using this method minimizes the amount of protein material used.
From previous work8, it is apparent that during the crystal growth process, in situ observations need to be carried out at regular time intervals. These can range from a few seconds to several days, depending on the event under observation (precipitation, nucleation, or crystal growth).
The optimization of crystal growth with OptiCrys is based on temperature-precipitant concentration phase diagrams. In the case of proteins with solubility as a direct function of temperature, it is possible to make use of the salting-out regime18. This is where increasing the ionic strength of the solution, which can be visualized using protein-precipitant phase diagrams, decreases the solubility of the protein. Likewise, proteins with inverse solubility can make use of the salting-in regime18. Nucleation occurs in the nucleation zone, in the vicinity of the metastable zone, and crystal growth then takes place in the metastable zone of the phase diagram until the protein concentration reaches the solubility limit. As shown in Figure 3A, with constant chemical composition temperature can be decreased to keep the crystallization solution in the metastable zone to prevent new nucleation. Crystals grow until the second crystal/solution equilibrium is achieved and after that, no further increase in the size of crystals is observed. The temperature is decreased several times until the crystals reach the desired size. In Figure 3B, at constant temperature, increasing the precipitant concentration keeps the solution in the metastable zone. This process can then be repeated several times to obtain large crystals. Changing the temperature and manipulating the crystallization solution conditions, by controlling the supersaturation levels, are two powerful tools for separating nucleation and growth of crystals that are controlled precisely and automatically by OptiCrys5,8,14.
Examples of protein crystals grown by temperature-controlled, or temperature- and precipitant concentration-controlled crystallization, as well as relative diffraction data obtained are available in the literature and PDB. Among them are human &#947;-crystallin E, PA-IIL lectin, yeast inorganic pyrophosphatase, urate oxidase, human carbonic anhydrase II, YchB kinase, and lactate dehydrogenase5,14,17,18.
Although OptiCrys was commercialized by NatX-ray, there are many laboratories that do not have access to this instrument or to the serial approach it offers. The alternative to this technique is to use commercially available plastic microdialysis buttons with various volumes. Using these, temperature and chemical composition can be adjusted and varied manually. Inspection of microdialysis buttons cannot be done in situ and must instead be done manually with an optical microscope. Temperature control can be achieved by keeping the sample in a vibration-free temperature-controlled incubator. It is essential to keep the temperature constant to ensure that crystallization experiments are reproducible. Significant variation in temperature may also lead to damage or destruction of crystals5.
Here we provide a detailed protocol describing sample preparation and the use of control software for the growth of large, high-quality crystals suitable for neutron protein crystallography. This step-by-step procedure was designed to take advantage of the crystallization phase diagram in order to select a starting position and kinetic path to control the size and the quality of the crystals generated. Additionally, a detailed protocol for growing crystals with microdialysis buttons is presented which uses the same rationale to obtain large, high-quality crystals.

<table>
	<tbody>
		<tr>
			<td>200 &#181;l Dialysis Button</td>
			<td>Hampton Research</td>
			<td>HR3-330</td>
			<td>Dialysis button</td>
		</tr>
		<tr>
			<td>24 well plates</td>
			<td>Jena Bioscience</td>
			<td>CPL-132</td>
			<td>Crystallization plate</td>
		</tr>
		<tr>
			<td>2-Switch</td>
			<td>FLUIGENT</td>
			<td>2SW001</td>
			<td>Switch</td>
		</tr>
		<tr>
			<td>30 &#956;l Dialysis Button</td>
			<td>Hampton Research</td>
			<td>HR3-324</td>
			<td>Dialysis button</td>
		</tr>
		<tr>
			<td>50 mL Corning Centrifuge tubes</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430828-500EA</td>
			<td>Centrifuge tubes</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>S2889</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Chicken Egg White Lysozyme</td>
			<td>Sigma-Aldrich</td>
			<td>L6876</td>
			<td>Lyophilized protein powder</td>
		</tr>
		<tr>
			<td>Dialysis Membrane Discs 6-8 kDa MWCO</td>
			<td>Spectrum</td>
			<td>132478</td>
			<td>Dialysis membrane</td>
		</tr>
		<tr>
			<td>Dialysis Membrane Tubing 6-8 kDa MWCO</td>
			<td>Spectrum</td>
			<td>132650T</td>
			<td>Dialysis membrane</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>Minispin</td>
			<td>Bench-top centrifuge</td>
		</tr>
		<tr>
			<td>Flow Unit</td>
			<td>FLUIGENT</td>
			<td>FLU-XL</td>
			<td>Flow meter</td>
		</tr>
		<tr>
			<td>Flowboard</td>
			<td>FLUIGENT</td>
			<td>FLB</td>
			<td>Flowboard</td>
		</tr>
		<tr>
			<td>Microfluidic Flow Control System EZ</td>
			<td>FLUIGENT</td>
			<td>EZ-01000002</td>
			<td>Pressure/vacuum controller</td>
		</tr>
		<tr>
			<td>MilliporeSigma 0.22 &#181;m syringe Filters</td>
			<td>Millipore</td>
			<td>GSWP04700</td>
			<td>0.22 &#956;m pore size filter</td>
		</tr>
		<tr>
			<td>M-Switch</td>
			<td>FLUIGENT</td>
			<td>MSW002</td>
			<td>Rotary valve</td>
		</tr>
		<tr>
			<td>Opticrys</td>
			<td>NatX-ray</td>
			<td>PRT008</td>
			<td>Crystallization bench</td>
		</tr>
		<tr>
			<td>Siliconized circle cover slides</td>
			<td>Hampton Research</td>
			<td>HR3-231</td>
			<td>Glass slides</td>
		</tr>
		<tr>
			<td>Sodium Chloride &#8805; 99%</td>
			<td>Sigma-Aldrich</td>
			<td>746398</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Switchboard</td>
			<td>FLUIGENT</td>
			<td>SWB002</td>
			<td>Switchboard</td>
		</tr>
		<tr>
			<td>Thermoregulated incubator</td>
			<td>Memmert</td>
			<td>IPP30</td>
			<td>Thermoregulated incubator</td>
		</tr>
	</tbody>
</table>

optimization of crystal growth for neutron macromolecular crystallography

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX beamlines having been built and increased availability of structure refinement software. However, the neutron sources currently available for NMX are significantly weaker than equivalent sources for X-ray crystallography. Despite advances in this field, significantly larger crystals will always be required for neutron diffraction studies, particularly with the tendency to study ever-larger macromolecules and complexes. Further improvements in methods and instrumentation suited to growing larger crystals are therefore necessary for the use of NMX to expand.
In this work, we introduce rational strategies and a crystal growth bench (OptiCrys) developed in our laboratory that combines real-time observation through a microscope-mounted video camera with precise automated control of crystallization solutions (e.g., precipitant concentration, pH, additive, temperature). We then demonstrate how this control of temperature and chemical composition facilitates the search for optimal crystallization conditions using model soluble proteins. Thorough knowledge of the crystallization phase diagram is crucial for selecting the starting position and the kinetic path for any crystallization experiment. We show how a rational approach can control the size and number of crystals generated based on knowledge of multidimensional phase diagrams.

In Sections 2.3 and 2.4, three examples of optimized crystal growth are presented, showing use of the instrument and an experimental design for growing large crystals. For this demonstration, we have used lysozyme as a model protein, although crystal growth experiments have been successfully performed with many other protein systems using this method (see above). By using and mastering the protocol presented here one can adapt it for other protein candidates.
In section 2.3 we demonstrated that established rational crystallization strategies could be beneficial in growing crystals with sufficient scattering volumes for neutron protein crystallography. Here, we demonstrate that the rational optimization strategies proposed also allow the generation of a uniform population of crystals of any specific size required for downstream structure determination approaches.
These two experiments are designed to emphasize the importance of phase diagrams in controlling crystal nucleation and growth. Here, control of the temperature and chemical composition of crystallization solutions in combination with monitoring the crystallization process in real time are used to study the qualitative phase diagram. Using this method, nucleation and crystal growth can be rationally optimized in a reversible manner. Use of such a serial approach also reduces the amount of protein and the time required to control the size and quality of the crystals.
In the dialysis method, a protein solution is separated from a crystallization solution by a semi-permeable membrane6 (Figure 5). This dialysis membrane allows small molecules such as additives, buffer, and ions to pass through the membrane but not macromolecules such as proteins6,20. This feature allows the crystallization solution to be modified during the course of the experiment6. Exchange of the solution can be done manually, for example in microdialysis buttons, or in an automated manner using an instrument developed for this purpose, OptiCrys8.
In the first set of experiments, microdialysis buttons were used for the crystallization of chicken egg-white lysozyme. Microdialysis buttons were immersed in crystallization solutions with different salt concentrations. In this simple crystallization grid experiment, the only variable is precipitant concentration whilst temperature is kept constant (293 K). As shown in Figure 4, slight variations in the salt concentration induce a change in the size and numbers of crystals observed, allowing investigation of the crystallization phase diagram. In Figure 4, panel 1, the crystallization solution contains 0.7 M NaCl and a limited number of larger crystals have appeared in the buttons. By increasing salt concentration from 0.7 to 1.2 M, supersaturation increases and the solution in the nucleation zone moves away from the metastable zone (Figure 4, panels 1 to 6). As a result, the number of crystals increases and their size decreases.
In the first experiment with a fully automated instrument enabling temperature-controlled dialysis crystallization, OptiCrys (Figure 9), the crystal growth experiment was tailored to generate large crystal growth. The experiment was launched at an initial temperature of 295 K with a crystallization solution containing 0.75 M NaCl and 0.1 M Na acetate buffer pH 4. Under these experimental conditions, the crystallization solution reached the nucleation zone in the vicinity of the metastable zone of the phase diagram (Figure 9, arrow 1). As a result, only a few nuclei were generated during the first stage of the experiment. In order to grow selected crystals further (shown in Figure 9), the crystal growth optimization workflow was driven towards the metastable zone by varying temperature as soon as the crystal-solution equilibrium was reached.
Each time equilibrium between crystal and solution was reached, the temperature was lowered, first to 291 K, then to 288 K and finally to 275 K, to keep the crystallization solution in the metastable zone. The result of this experiment is a single large crystal suitable for both macromolecular X-ray and neutron crystallography.
For most proteins, the precise quantitative phase diagram (or just a qualitative diagram) has not yet been obtained due to the lack of experimental devices capable of accurately measuring protein concentration (or just of observing/detecting the crystallization process in real time) during crystallization experiments18. As a result, it is often not possible to design the experiment in such a way that crystallization begins in the optimal area of the phase diagram, in the vicinity of the metastable zone.
Therefore, a crystallization optimization study must take place before the experiment dedicated to the growth of a large volume crystal is undertaken. In this study, using temperature variations (at constant chemical composition) on the one hand and variations in chemical composition (at constant temperature) on the other hand, are&#160;necessary to identify the metastable zone and to delineate the optimal conditions for starting a large crystal growth experiment.
To this end, two other experiments are presented which were tailored to demonstrate the reversibility of the temperature-controlled dialysis crystallization experiments with OptiCrys for nucleation, crystal growth, dissolution and re-growth. The crystal growth optimization workflow was controlled so that a uniform population of fewer, larger lysozyme crystals was grown, using variation of temperature or precipitant concentration.
In the second experiment with OptiCrys, the chemical composition of the crystallization solution was kept constant throughout the experiment (0.9 M NaCl in 0.1 M CH3COONa pH 4) with variable temperature. The initial temperature was set at 291 K. The results of this experiment are summarized in Figure 10. Because of high supersaturation, a large number of small crystals appeared in the crystallization chamber (Figure 10, panels 1 and 2). In accordance with the concept of direct protein solubility, by gradually increasing the temperature to 313 K, all of the crystals were dissolved (Figure 10, panels 3, 4 and 5). Finally, by lowering the temperature to 295 K, the second nucleation was initiated in the vicinity of the metastable zone and allowed controlled formation of a lower number of nuclei. Further crystal growth resulted in the uniform generation of a population of larger crystals (Figure 10, panel 7).
As shown in Figure 11, variation of the chemical composition of the crystallization solution, at a constant temperature of 291 K, can likewise be used to obtain a uniform population of larger crystals. Similar to the previous experiment, the initial condition was 0.9 M NaCl in 0.1 M CH3COONa pH 4. The NaCl concentration was then lowered gradually from 0.9 M to zero to dissolve the crystals (Figure 11, panels 4 and 5). At this point, NaCl was completely replaced by a buffer solution of 0.1 M CH3COONa pH 4. Reducing the salt concentration keeps the solution in the undersaturated zone of the phase diagram, which leads to the dissolution of the crystals. Then, a new crystallization solution with lower ionic strength, at 0.75 M NaCl in 0.1 M CH3COONa pH 4, was injected into the reservoir chamber. At this precipitant concentration, the first nuclei appeared (Figure 11, panel 6) after 90 minutes. The number of generated crystals was lower and the crystals reach a larger volume (Figure 11, panel 7) than before.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61685/61685fig1v3.jpg" /> 
Figure 1: Schematic phase diagram.&#160;Kinetic trajectories for three crystallization techniques are represented in a salting-out regime. Each method achieves nucleation and crystallization differently, visualized by a different kinetic pathway through the phase diagram to reach the nucleation and metastable zones. The solubility curve separates undersaturation and supersaturation regions. Supersaturation is divided into three zones: metastable, nucleation and precipitation. In the nucleation zone, spontaneous nucleation occurs while in the metastable zone crystal growth takes place. This Figure is adapted from Junius et al.8 <a href="https://www.jove.com/files/ftp_upload/61685/61685fig1v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61685/61685fig02.jpg" /> 
Figure 2: Schematic representation of the crystallization bench (OptiCrys).&#160;The LED light source is located on top of the temperature-controlled dialysis flow cell. An inverted microscope and the digital camera are shown at the top right of the image with the red arrow. The red circle represents the location of the chiller tubing. <a href="https://www.jove.com/files/ftp_upload/61685/61685fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61685/61685fig03.jpg" /> 
Figure 3: Schematic two-dimensional protein crystallization phase diagram as a function of temperature (A) and precipitant concentration (B).&#160;(A) In case of a protein with direct solubility, decreasing the temperature keeps the crystallization solution in the metastable zone. Temperature variation can be repeated several times to control the crystal growth process until crystals with the desired volume are obtained. (B) Changing the concentration of the precipitant solution can also be used to keep the crystallization solution in the metastable zone for growing crystals. This Figure is adapted from Junius et al.8 <a href="https://www.jove.com/files/ftp_upload/61685/61685fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61685/61685fig04.jpg" /> 
Figure 4: Crystals of lysozyme obtained using the dialysis method.&#160;This experiment was performed at a constant temperature of 293 K in 0.1 M sodium acetate buffer pH 4. Increasing NaCl concentration from 0.7 M to 1.2 M increases the nucleation rate and results in a larger number of crystals. <a href="https://www.jove.com/files/ftp_upload/61685/61685fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61685/61685fig05.jpg" /> 
Figure 5: Overview of the protein crystallization process by the dialysis method.&#160;(A) By adding the protein to the chamber of the dialysis button, (B) a dome shape is created on the top of the chamber. (C) An applicator is used to transfer the O-ring to the groove of the dialysis button&#160;in order to fix the dialysis membrane in place. (D) The dialysis button is ready for immersion in the reservoir solution. (E) Crystallization solution passes through the semipermeable membrane and crystals start to form inside the chamber. <a href="https://www.jove.com/files/ftp_upload/61685/61685fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61685/61685fig06.jpg" /> 
Figure 6: Schematic view of the temperature-controlled flowing dialysis setup.&#160;(A) The protein sample is added to the dialysis chamber. (B) The dialysis membrane is fixed onto the overchamber with an O-ring by using an applicator. (C) The overchamber is turned and fixed onto the top of the dialysis chamber. White arrows indicate where&#160;screws are&#160;placed on the overchamber. (D) The reservoir chamber is turned clockwise (E) and fixed on top of the overchamber. (F) The reservoir chamber is covered by an airtight cap with connectors to a pumping system and (G) the flow cell is placed in the brass support. This Figure is adapted from Junius et al.8 <a href="https://www.jove.com/files/ftp_upload/61685/61685fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61685/61685fig07.jpg" /> 
Figure 7: Preparation and injection of the crystallization solution in the reservoir by the fluidic system (A).&#160;Tubes containing salt and water are connected to the pressure/vacuum controller (B) and to the rotary valve (C). By using the pressure, pressure/vacuum controller creates a constant flow of the liquids from the tubes to the rotary valve. Each liquid passing through the flow meter (D) and the switch is injected into the mixing tube (F). Once all the liquids have been added to the mixing tube, the switch by some modifications injects the final solution from the mixing tube into the reservoir (G). The liquid flows through the system in the direction of the arrows in the diagram marked in ascending order (from 1 to 6). <a href="https://www.jove.com/files/ftp_upload/61685/61685fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61685/61685fig8v2.jpg" /> 
Figure 8: Maintenance view of the supervision software.&#160;This view is used to control different parameters like temperature, light, crystallization solution and zoom. <a href="https://www.jove.com/files/ftp_upload/61685/61685fig8v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/61685/61685fig09.jpg" /> 
Figure 9: The phase diagram as a function of the temperature (selected images are to be tracked in ascending order).&#160;A single large lysozyme crystal is obtained by systematically changing the temperature from 295 K to 275 K. At each step, crystal growth is stopped upon reaching the solubility curve. Reducing the temperature by keeping the solution in the metastable zone restarts crystal growth. The images have different levels of magnification. This Figure is adapted from Junius et al.8,18 <a href="https://www.jove.com/files/ftp_upload/61685/61685fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/61685/61685fig10.jpg" /> 
Figure 10: Optimization of crystal growth at constant chemical composition using temperature control (selected images are to be tracked in ascending order).&#160;Starting the nucleation process in the nucleation zone at 291 K, far from the metastable zone, results in the formation of numerous crystals. Increasing the temperature to 313 K then dissolves the crystals until no visible nuclei are seen in the dialysis chamber. Finally, decreasing the temperature to 295 K restarts the nucleation process for the second time leading to a limited number of larger crystals. This Figure is adapted from Junius et al.8,18 <a href="https://www.jove.com/files/ftp_upload/61685/61685fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/61685/61685fig11.jpg" /> 
Figure 11: Optimization of crystal growth at constant temperature using variations in precipitant concentration (selected images are to be tracked in ascending order).&#160;Decreasing the precipitant concentration from 0.9 M to 0 M dissolves the crystals obtained during the first nucleation event. The crystallization process is restarted by the injection of the same precipitant but at lower ionic strength, 0.75 M, which leads to the formation of a few larger crystals. This Figure is adapted from Junius et al.8,18 <a href="https://www.jove.com/files/ftp_upload/61685/61685fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Optimization of Crystal Growth for Neutron Macromolecular Crystallography at JoVE.com

Optimization of Crystal Growth for Neutron Macromolecular Crystallography

Structural studies of biomacromolecules by crystallography require high-quality crystals. Here we demonstrate a protocol that can be used by OptiCrys (a fully automated instrument developed in our lab) and/or microdialysis buttons for growing large high-quality crystals based on knowledge of the crystallization phase diagram.

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX ...

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5.3K Views.  Université Grenoble Alpes. Structural studies of biomacromolecules by crystallography require high-quality crystals. Here we demonstrate a protocol that can be used by OptiCrys (a fully automated instrument developed in our lab) and/or microdialysis buttons for growing large high-quality crystals based on knowledge of the crystallization phase diagram.

Video: Optimization of Crystal Growth for Neutron Macromolecular Crystallography

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Protein Crystallization for X-ray Crystallography

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

Isolation and Biophysical Study of Fruit Cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment.1 A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer 'epicuticular' layer as well as the blend consisting of the cutin biopolymer and 'intracuticular' waxes.2 Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato (Solanum lycopersicum) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Sch&auml;ffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle.3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) 13C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS 13C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH2O) chemical moieties.
Exhaustive dewaxing by stepwise Soxhlet extraction with a panel of solvents of varying polarity provides an effective means to isolate wax moieties based on the hydrophobic characteristics of their aliphatic and aromatic constituents, while preserving the chemical structure of the cutin biopolyester. The mechanical extraction of epicuticular waxes and selective removal of intracuticular waxes, when monitored by complementary physical methodologies, provides an unprecedented means to investigate the cuticle assembly: this approach reveals the supramolecular organization and structural integration of various types of waxes, the architecture of the cutin-wax matrix, and the chemical composition of each constituent. In addition, solid-state 13C NMR reveals differences in the relative numbers of CHO and CH2O chemical moieties for wild-type and mutant red ripe tomato fruits. The NMR techniques offer exceptional tools to fingerprint the molecular structure of cuticular materials that are insoluble, amorphous, and chemically heterogeneous. As a noninvasive surface-selective imaging technique, AFM furnishes an effective and direct means to probe the structural organization of the cuticular assembly on the nm-&mu;m length scale.

Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters within the origin of replication1. Beyond the origin, SeqA is found at the replication forks, where it organizes newly replicated DNA into higher ordered structures2. SeqA associates only weakly with single GATC sequences, but it forms high affinity complexes with DNA duplexes containing multiple GATC sites. The minimal functional and structural unit of SeqA is a dimer, thereby explaining the requirement of at least two GATC sequences to form a high-affinity complex with hemimethylated DNA3. Additionally, the SeqA architecture, with the oligomerization and DNA-binding domains separated by a flexible linker, allows binding to GATC repeats separated by up to three helical turns. Therefore, understanding the function of SeqA at a molecular level requires the structural analysis of SeqA bound to multiple GATC sequences. In protein-DNA crystallization, DNA can have none to an exceptional effect on the packing interactions depending on the relative sizes and architecture of the protein and the DNA. If the protein is larger than the DNA or footprints most of the DNA, the crystal packing is primarily mediated by protein-protein interactions. Conversely, when the protein is the same size or smaller than the DNA or it only covers a fraction of the DNA, DNA-DNA and DNA-protein interactions dominate crystal packing. Therefore, crystallization of protein-DNA complexes requires the systematic screening of DNA length4 and DNA ends (blunt or overhang)5-7. In this report, we describe how to design, optimize, purify and crystallize hemimethylated DNA duplexes containing tandem GATC repeats in complex with a dimeric variant of SeqA (SeqA&Delta;(41-59)-A25R) to obtain crystals suitable for structure determination.

Crystal structure of protein&#x2013;DNA complexes can provide insight into protein function, mechanism, as well as, the nature of the specific interaction. Here, we report how to optimize the length, sequence and ends of duplex DNA for co-crystallization with Escherichia coli SeqA, a negative regulator of replication initiation.

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. The S domain forms a contiguous scaffold around the viral RNA, whereas the P domain forms viral spikes on the S domain and contains determinants for antigenicity and host-cell interactions. The P domain binds carbohydrate structures, i.e., histo-blood group antigens, which are thought to be important for norovirus infections. In this protocol, we describe a method for producing high quality norovirus P domains in high yields. These proteins can then be used for X-ray crystallography and ELISA in order to study antigenicity and host-cell interactions.
The P domain is firstly cloned into an expression vector and then expressed in bacteria. The protein is purified using three steps that involve immobilized metal-ion affinity chromatography and size exclusion chromatography. In principle, it is possible to clone, express, purify, and crystallize proteins in less than four weeks, which makes this protocol a rapid system for analyzing newly emerging norovirus strains.

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Here, we describe a method to express and purify high quality norovirus protruding (P) domains in E. coli for use in X-ray crystallography studies. This method can be applied to other calicivirus P domains, as well as non-structural proteins, i.e., viral protein genome-linked (VPg), protease, and RNA dependent RNA polymerase (RdRp).

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. ...

The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid acquisition require assessing mouthpart structure and function relationships; however, fluid uptake mechanisms are historically inferred from observations of structural architecture, sometimes unaccompanied with experimental evidence. Here, we report a novel method for assessing fluid-uptake abilities with butterflies (Lepidoptera) and flies (Diptera) using small amounts of liquids. Insects are fed with a 20% sucrose solution mixed with fluorescent, magnetic nanoparticles from filter papers of specific pore sizes. The crop (internal structure used for storing fluids) is removed from the insect and placed on a confocal microscope. A magnet is waved by the crop to determine the presence of nanoparticles, which indicate if the insects are able to ingest fluids. This methodology is used to reveal a widespread feeding mechanism (capillary action and liquid bridge formation) that is potentially shared among Lepidoptera and Diptera when feeding from porous surfaces. In addition, this method can be used for studies of feeding mechanisms among a variety of fluid-feeding insects, including those important in disease transmission and biomimetics, and potentially other studies that involve nano- or micro-sized conduits where liquid transport requires verification.

Fluid-feeding insects have the ability to acquire minute quantities of liquids from porous surfaces. This protocol describes a method to directly determine the ability for insects to ingest liquids from porous surfaces using feeding solutions with fluorescent, magnetic nanoparticles.

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...