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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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 &amp;micro;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 &amp;mu;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 &amp;micro;m syringe Filters</td><td>Millipore</td><td>GSWP04700</td><td>0.22 &amp;mu;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 &amp;ge; 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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Biology

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

Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology

When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution. The conditions to crystallize proteins, DNAs, RNAs, and their complexes can however not be predicted. Employing a broad variety of conditions is a way to increase the yield of quality diffraction crystals. Two fully automated systems have been developed at the MRC Laboratory of Molecular Biology (Cambridge, England, MRC-LMB) that facilitate crystallization screening against 1,920 initial conditions by vapor diffusion in nanoliter droplets. Semi-automated protocols have also been developed to optimize conditions by changing the concentrations of reagents, the pH, or by introducing additives that potentially enhance properties of the resulting crystals. All the corresponding protocols will be described in detail and briefly discussed. Taken together, they enable convenient and highly efficient macromolecular crystallization in a multi-user facility, while giving the users control over key parameters of their experiments.

Automated systems and protocols for the routine preparation of a large number of screens and nanoliter crystallization droplets for vapor diffusion experiments are described and discussed.

When high quality crystals are obtained that diffract X-rays, the crystal structure may be solved at near atomic resolution. The conditions to crystallize ...

Biochemistry

Microcrystallography of Protein Crystals and In Cellulo Diffraction

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in their largest dimension, which used to represent a challenge. We present two alternative workflows for the structure determination of protein microcrystals by X-ray crystallography with a particular focus on crystals grown in vivo. The microcrystals are either extracted from cells by sonication and purified by differential centrifugation, or analyzed in cellulo after cell sorting by flow cytometry of crystal-containing cells. Optionally, purified crystals or crystal-containing cells are soaked in heavy atom solutions for experimental phasing. These samples are then prepared for diffraction experiments in a similar way by application onto a micromesh support and flash cooling in liquid nitrogen. We briefly describe and compare serial diffraction experiments of isolated microcrystals and crystal-containing cells using a microfocus synchrotron beamline to produce datasets suitable for phasing, model building and refinement.
These workflows are exemplified with crystals of the Bombyx mori cypovirus 1 (BmCPV1) polyhedrin produced by infection of insect cells with a recombinant baculovirus. In this case study, in cellulo analysis is more efficient than analysis of purified crystals and yields a structure in ~8 days from expression to refinement.

Microcrystallography of Protein Crystals and In Cellulo Diffraction

A protocol is presented for X-ray crystallography using protein microcrystals. Two examples analyzing in vivo-grown microcrystals after purification or in cellulo are compared.

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in ...

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for even the most challenging projects in structural biology. However, most facilities still require the presence of a scientist on site to perform the experiments. A new generation of automated beamlines dedicated to the fully automatic characterization of, and data collection from, crystals of biological macromolecules has recently been developed. These beamlines represent a new tool for structural biologists to screen the results of initial crystallization trials and/or the collection of large numbers of diffraction data sets, without users having to control the beamline themselves. Here we show how to set up an experiment for automatic screening and data collection, how an experiment is performed at the beamline, how the resulting data sets are processed, and how, when possible, the crystal structure of the biological macromolecule is solved.

Here, we describe how to use the automated screening and data collection options available at some synchrotron beamlines. Scientists send cryocooled samples to the synchrotron, and the diffraction properties are screened, the data sets are collected and processed and, where possible, a structure solution is carried out&#8212;all without human intervention.

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

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

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

A novel sample holder for macromolecular X-ray crystallography along with a suitable handling protocol is presented. The system allows crystal growth, crystal soaking and in situ diffraction data collection at both, ambient and cryogenic temperature without the need of any crystal manipulation or mounting.

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. ...

Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial crystallography experiments at both synchrotrons and XFELs. The method is based upon an understanding of the protein crystal phase diagram, and how that knowledge can be utilized. The method is divided into three stages: (1) optimizing crystal morphology, (2) transitioning to batch, and (3) scaling. Stage 1 involves finding well diffracting, single crystals, hopefully but not necessarily, presenting in a cube-like morphology. In Stage 2, the Stage 1 condition is optimized by crystal growth time. This strategy can transform crystals grown by vapor diffusion to batch. Once crystal growth can occur within approximately 24 h, a morphogram of the protein and precipitant mixture can be plotted and used as the basis for a scaling strategy (Stage 3). When crystals can be grown in batch, scaling can be attempted, and the crystal size and concentration optimized as the volume is increased. Endothiapepsin has been used as a demonstration protein for this protocol. Some of the decisions presented are specific to endothiapepsin. However, it is hoped that the way they have been applied will inspire a way of thinking about this procedure that others can adapt to their own projects.

The aim of this article is to give the viewer a solid understanding of how to transform their small-volume, vapor-diffusion protocol, for growing large, single protein crystals, into a large-volume batch micro-crystallization method for serial crystallography.

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial ...

A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality have been seen for microcrystals with the development of beamline optics, beam stability and variable beam size focusing from submicron to microns, such as at the VMXm beamline at Diamond Light Source1. Further improvements in data quality will be gained through improvements in sample environment and sample preparation. Microcrystals inherently generate weaker diffraction, therefore improving the signal-to-noise is key to collecting quality X-ray diffraction data and will predominantly come from reductions in background noise. Major sources of X-ray background noise in a diffraction experiment are from their interaction with the air path before and after the sample, excess crystallization solution surrounding the sample, the presence of crystalline ice and scatter from any other beamline instrumentation or X-ray windows. The VMXm beamline comprises instrumentation and a sample preparation protocol to reduce all these sources of noise.
Firstly, an in-vacuum sample environment at VMXm removes the air path between X-ray source and sample. Next, sample preparation protocols for macromolecular crystallography at VMXm utilize a number of processes and tools adapted from cryoTEM. These include copper grids with holey carbon support films, automated blotting and plunge cooling robotics making use of liquid ethane. These tools enable the preparation of hundreds of microcrystals on a single cryoTEM grid with minimal surrounding liquid on a low-noise support. They also minimize the formation of crystalline ice from any remaining liquid surrounding the crystals. 
We present the process for preparing and assessing the quality of soluble protein microcrystals using visible light and scanning electron microscopy before mounting the samples on the VMXm beamline for X-ray diffraction experiments. We will also provide examples of good quality samples as well as those which require further optimization and strategies to do so.

The signal-to-noise ratio of data is one of the most important considerations in performing X-ray diffraction measurements from microcrystals. The VMXm beamline provides a low-noise environment and microbeam for such experiments. Here, we describe sample preparation methods for mounting and cooling microcrystals for VMXm and other microfocus macromolecular crystallography beamlines.

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality ...

Neutron Crystallography Data Collection and Processing for Modelling Hydrogen Atoms in Protein Structures

Neutron crystallography is a structural technique that allows determination of hydrogen atom positions within biological macromolecules, yielding mechanistically important information about protonation and hydration states while not inducing radiation damage. X-ray diffraction, in contrast, provides only limited information on the position of light atoms and the X-ray beam&#160;rapidly induces radiation damage of photosensitive&#160;cofactors and metal centers. Presented here is the workflow employed for the IMAGINE and MaNDi beamlines at Oak Ridge National Laboratory (ORNL) to obtain a neutron diffraction structure once a protein crystal of suitable size (&#62; 0.1 mm3) has been grown. We demonstrate mounting of hydrogenated protein&#160;crystals in quartz capillaries for neutron diffraction data collection. Also presented is the vapor exchange process of the mounted crystals with D2O-containing buffer to ensure replacement of hydrogen atoms at exchangeable sites with deuterium. The incorporation of deuterium reduces the background arising from the incoherent scattering of hydrogen atoms and prevents density cancellation caused by their negative coherent scattering length. Sample alignment and room temperature data collection strategies are illustrated using quasi-Laue data collection at IMAGINE at the High Flux Isotope Reactor (HFIR). Furthermore, crystal mounting and rapid freezing in liquid nitrogen for cryo-data collection to trap labile reaction intermediates is demonstrated at the MaNDi time-of-flight instrument at the Spallation Neutron Source (SNS). Preparation of the model coordinate and diffraction data files and visualization of the neutron scattering length density (SLD) maps will also be addressed. Structure refinement against neutron data-only or against joint X-ray/neutron data to obtain an all-atom structure of the protein of interest will finally be discussed. The process of determining a neutron structure will be demonstrated using crystals of the lytic polysaccharide monooxygenase Neurospora crassa LPMO9D, a copper-containing metalloprotein involved in the degradation of recalcitrant polysaccharides via oxidative cleavage of the glycosidic bond.

Neutron protein crystallography is a structural technique that permits the localization of hydrogen atoms, thereby providing important mechanistic details of protein function. We present here the workflow for mounting a protein crystal, neutron diffraction data collection, structure refinement and analysis of the neutron scattering length density maps.

Neutron crystallography is a structural technique that allows determination of hydrogen atom positions within biological macromolecules, yielding ...

Chemistry

High-Throughput Protein Crystallization via Microdialysis

Understanding the structure-function relationships of macromolecules, such as proteins, at the molecular level is vital for biomedicine and modern drug discovery. To date, X-ray crystallography remains the most successful method for solving three-dimensional protein structures at atomic resolution. With recent advances in serial crystallography, either using X-ray free electron lasers (XFELs) or synchrotron light sources, protein crystallography has progressed to the next frontier, where the ability to acquire time-resolved data provides important mechanistic insights into the behavior of biological molecules at room temperature. This protocol describes a straightforward high-throughput (HTP) workflow for screening crystallization conditions through the use of a 96-well dialysis plate. These plates follow the Society for Biomolecular Screening (SBS) standard and can be easily set up using any standard crystallization laboratory. Once optimal conditions are identified, large quantities of crystals (hundreds of microcrystals) can be produced using the dialyzer. To validate the robustness and versatility of this approach, four different proteins were crystallized, including two membrane proteins.

High-Throughput Protein Crystallization via Microdialysis

The presented protocol describes a straightforward approach for screening protein crystallization conditions and crystal growth using a 96-well high-throughput dialysis plate. The use of dialyzer tubes for the large-scale growth of microcrystals is also demonstrated for serial crystallography and MicroED applications.

Understanding the structure-function relationships of macromolecules, such as proteins, at the molecular level is vital for biomedicine and modern drug ...

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 ...

Optimization of Crystal Growth for Neutron Macromolecular Crystallography

Summary

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Chapters in this video

Automated Protocols for Macromolecular Crystallization at the MRC Laboratory of Molecular Biology

Microcrystallography of Protein Crystals and In Cellulo Diffraction

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

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

Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments

A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline

Neutron Crystallography Data Collection and Processing for Modelling Hydrogen Atoms in Protein Structures

High-Throughput Protein Crystallization via Microdialysis

Protein Crystallization for X-ray Crystallography