Optimization of Crystal Growth for Neutron Macromolecular Crystallography

There are different crystallization approaches for obtaining large and high quality crystals for both X-ray and neutron crystallography. One of these approaches is using dialysis method. In this technique, the protein solution is separated from a precipitant solution by a semi-permeable membrane.

The precipitant molecules diffuse across the membrane into the protein chamber. When the correct supersaturation for a spontaneous nucleation is achieved, the first nuclei will appear. Here, we will demonstrate a protocol that can be used by OptiCrys a fully automated instrument developed in our lab that uses temperature controlled dialysis crystallization, and our micro dialysis buttons for growing large high quality crystals for natural macromolecular crystallography based on knowledge of the crystallization phase diagram.

For a micro dialysis button with certain microliter volume pipette 35 microliters of lysozyme solution into the dialysis chamber. This extra volume creates a slightly dome shape on top of the dialysis chamber and prevents air bubble formation. Take an applicator and place the elastic or ring at the extremity of it.

Place the membrane on top of the chamber and fix it by transferring the elastic or ring from the applicator to the groove of the dialysis button. Transfer the button to the well containing the crystallization solution. Cover the well with a glass cover slip or a piece of tape.

Keep the sample at 293 Kelvin in a vibration free thermal regulator incubator. Knowing that the dialysis crystallization offers advantages from the point of view of crystal growth the crystallization bench so-called OptiCrys was designed and developed in our laboratory to offer a fully automated combined control of temperature and chemical composition of the biomolecular crystallization experiment. The optimization of the crystal growth by OptiCrys is based on the temperature precipitant concentration phase diagrams.

Nucleation occurs in the nucleation zone in the vicinity of the metastable zone and crystal growth then take place in the metastable zone after phase diagram, until the protein concentration reaches the solubility curve representing the thermodynamical equilibrium between solution and crystals. By decreasing the temperature in case of the protein with direct solubility or by increasing the precipitant concentration in case of salting out reaching, the crystallization solution remains in the metastable zone and no nucleation occurs. Crystals grow until the second crystal solution equilibrium achieve and after that, no further increase in the size of crystals is observed.

Decreasing the temperature or increasing the precipitant concentration is repeated several times until the crystals reach the desired size. Add the protein solution to the dialysis chamber of the temperature controlled flowing set up, cover the over chamber with the dialysis membrane and fix the membrane with the elastic or ring. Flip the over chamber and put it on top of the dialysis chamber.

Press it gently and slowly to remove all the air trapped between two pieces, fix the reservoir in it's position by gently screwing it on top of the over chamber. Add the crystallization solution to the reservoir chamber and cover it by the airtight cap. Transfer this assembly and inserted it into the breast support of the crystallization bench OptiCrys Crystal growth is a supervision software of the Crystallization Bench OptiCrys.

It includes four different graphical interfaces or views. The home view contains buttons to navigate to the other views. The purpose of the setup interface is to define an experiment scenario that can be run automatically in the test view.

The last view is the maintenance view. To restart an experiment, the maintenance view must be open where all the essential parameters for the smooth running off the crystallization experiments are found. Increase the luminosity from the light section, light can increase from zero, no light to 100 maximum brightness.

For controlling and monitoring the temperature, the temperature regulator section is used. Click on the button to turn it on. Set the temperature on the set point section and press enter.

Below this button, there are two graphs. The red one shows the final, the order temperature and the yellow graph shows the current temperature. Mixing a stock solutions, an injection of the crystallization solution to the reservoir chamber is controlled by Palm section.

Add the concentration of the stock solution in the first stage. Then find the final concentration of each new grouper solution and add them to the final concentration section. After pressing the calculate button, find out volume of stock solution will be calculated and will add in the volume panel in front of each concentration panel.

Press the launch preparation button and wait until the new premier solution is ready. For exchanging the crystallization solution, click on the Solution Entry button. For stopping the process, press Stop Distribution button.

On the right side of the microscope section, There are several panels for recording important information from each crystal growth experiment. Add corresponding proteins name, molecular weight and crystallization condition in this section. Define a name for your experiments by simply typing it on the folder name.

From nb images section, select number of images that should be taken during the experiment. Add the number of the images and from the panel on the right, choose the timer scale. Click on the folder button to open the folder.

In this folder, there is a text file containing all the information you previously defined for your experiment. Images ready for any further treatment are also saved in this folder. Zoom off the microscope can also be varied automatically.

Changes your zoom by using the plus and minus button on top of the microscope section to increase or decrease the magnification respectively. There are three different ways to measure the crystal size. For measuring the length or width use the width vector.

Depending on the crystal shape, the specific tools from the left part of the microscope section can be chosen. Such as a rectangular or polygon. The values will appear in the measure section.

In the first set of experiments, microdialysis buttons were immersed in the crystallization solutions with different salt concentrations. In this simple crystallization grid experiment, the only variable is precipitant concentration. while the temperature is kept constant.

Slight variations in the salt concentration allow investigating the crystallization phase diagram by increasing salt concentration from 0.7 to 1.2 molar supersaturation increases and the solution in the nucleation zone is moving away from the metastable zone. Consequently, a variation in size and number of crystals is observed with the number of crystals increasing and their size decreasing. In the first experiment with OptiCrys under the given experimental condition, once the equilibrium in the dialysis chamber reach, the crystallization solution is in the nucleation zone in the vicinity of the metastable zone of crystallization phase diagram.

As a result, only a few nuclei are generated during the first stage of the experiment. In order to keep the growth of crystals in the metastable zone and control the crystal growth process, the temperature was changed at different time intervals. Each time the crystal solution equilibrium was completed, the temperature was varied.

Therefore the temperature decreased to 291 Kelvin, 288 Kelvin and finally 275 Kelvin to maintain the growth of selected crystal in the metastable zone. The result of this experiment is a single large crystal, with the volume required for natural macromolecular crystallography. Next two experiments demonstrate the reversibility of the temperature controlled dialysis experiments for nucleation, crystal growth, dissolution and regrowth.

In the second experiment with OptiCrys the chemical composition of the crystallization solution was kept constant through the experiment and the temperature varied. The initial temperature was set at 291 Kelvin. Because of high supersaturation, a large number of small crystals appeared in the crystallization chamber.

In accordance with the concept of direct protein solubility, by gradually increasing the temperature to 313 Kelvin all the crystals were to dissolved. Finally, by lowering the temperature to 295 Kelvin, the second nucleation initiated in the vicinity of the metastable zone and allowed to control the nucleation process resulting in forming of lower number of nuclei. Further crystal growth allowed generating the uniform population of larger crystals.

In this experiment, changing the chemical composition of the crystallization solution at constant temperature leads to obtain a uniform population of larger crystals. The crystallization condition was similar to the previous experiment. Dissolving the crystals was achieved by lowering gradually the NaCl concentration from 0.9 molar to zero.

Reducing the salt concentration keeps the solution in the undersaturated zone of the phase diagram which leads to the dissolution of the crystals. Then the new crystallization solution with lower ionic strength than previously was injected into the reservoir chamber. At this precipitant concentration the lower number of crystals appear and the crystals reach a larger volume than before.

Here we provided a detailed protocol describing sample preparation and adjusting the control software for growing large and high quality crystals for natural macromolecular crystallography using a fully automated instrument developed in our lab. This step-by-step procedure was designed and benefit from the knowledge of the crystallization phase diagram in order to separate nucleation and crystal growth. Moreover, a protocol for growing crystals with microcytosis patterns is also presented using macrocytosis buttons as an alternative when OptiCrys is not available.

In addition to the strategies that I mentioned for OptiCrys changing the chemical composition of the crystallization solution and temperature and taking images should be done manually. Using the thermal regulated vibration for the incubator to keep the temperature constant is required, which is a critical step in the methodology demonstrated.