This method can help to answer key questions in the protein crystallization field, such as time point of nucleation, mechanism of crystal growth or how to prepare nano/micro-crystals, as investigated by the Hamburg Center for Ultrafast Imaging. The main advantage of this technique is monitoring of sample evolution in real time, enabling the acquisition of valuable information, such as the radial distribution of particles in solution at different crystallization stages. Though this method can provide insight of protein micro-crystallization, it can also be used for proteins that are used for classic crystallography or for growth of big protein crystals to be used for neutron diffraction.

Visual demonstration of this method is important, as it shows the working principle of a unique crystallization device. To begin, filter 16 milliliters of 1.2 molar sodium tartrate solution and 16 milliliters of distilled water, using a 2 micron sterile syringe filter. Fill the precipitant and water bottles with five milliliters of the filtered solutions.

Then mount the bottles in the pump holders of the experimental chamber. In the software window, set up the experimental parameters with a temperature of 20 degrees Celsius, relative humidity of 20 and water as the solvent. Open the front door of the experimental chamber and remove the coverslip carrier.

Place a clean and siliconized coverslip on the carrier and place it back in the device. Close the experimental chamber to secure the environmental conditions. Switch on Pump Zero, using the main pump characteristics found in the text protocol and create a water stream.

Adjust the water stream, aiming the position towards the center of the coverslip by manually operating its designated adjustment screw. Switch off Pump Zero, then switch on Pump One and create a liquid stream, as before. Adjust the stream position of Pump One towards the position fixed for Pump Zero.

After switching off Pump One, replace the used coverslip with a new, clean coverslip for the crystallization experiment. Create a new experimental file using the software package. In the experimental file, enter the protein as Thaumatin, from Thaumatococcus daniellii;the protein concentration as 14 milligrams per milliliter, the precipitant as sodium tartrate and the precipitant concentration as 1.2 molar.

After loading the new experiment file to activate this information, mark the position on the protein droplet by adding a small water drop, using Pump Zero. Press the Tara'button to zero the microbalance. Open the top lid of the experimental chamber and pipette 8 microliters of Thaumatin solution on the water landmark.

To register the new Thaumatin drop, first click the New Drop'to attribute the initial conditions from the experimental file. Then click the Constant'button to compensate the natural evaporation of water from the droplet. Use the CCD camera to check if Pump Zero is aiming in the protein drop.

Readjust the position of Pump Zero if the water stream is aiming outside the drop. Switch on the DLS laser and place the laser beam in the protein drop by manually using the adjustment screws. Enter the DLS parameters with a measurement duration of 60 seconds, a waiting time between two measurements of 10 seconds and number of measurements as 300.

Click on the Start'button to initiate the DLS measurements. Check the quality of the sample by selecting one of the DLS graphic representations. Enter the conditions for the evaporation step in the schedule table, as shown in the text protocol.

Then activate the sample evaporation step by clicking the Automation'button. Click the Stop'button when the sample evaporation step has finished. Activate the constant button to keep the weight of the droplet constant.

This feature will compensate for the eventuality of natural sample evaporation and insure that no concentration changes can take place until further action is taken. Next, enter the conditions for the precipitant addition step in the schedule table, as listed in the text protocol. Activate the precipitant addition step by clicking the Automation'button.

To track the evolution of the crystallization droplet over time, check the appearance of the Thaumatin crystals by using the CCD camera. Check the particle size distribution by using the DLS graphic representations. Also check the evolution of weight and experimental parameters by using the display window.

All the information shown in this window are calculated and displayed in real time, based on the weight changes during crystallization, which are recorded online by the ultrasensitive balance. Coat a clean standard Terasaki plate by adding 3 milliliters of paraffin oil to the plate. Disburse the paraffin oil on the plate wells by gently moving the plate at different angles so that the oil covers all 72 wells.

Remove the excess paraffin oil by pouring out any oil that floats on the plate. Click the Stop'button to finish the crystallization experiment. Carefully take out the sample carrier containing the crystallization droplet.

With the use of a pipette, place two microliter aloquots of the crystallization drop in the wells of the Terasaki plate. By recovering the crystallization drop in a plate under oil, the sample can be periodically checked for stability in crystal growth with the use of a microscope or other diolist techniques that work with Terasaki plates. Displayed here is a radius plot distribution showing the growth of particle size in the protein drop during the entire crystallization process from monomeric protein toward formation of nucleii, nano-crystals and micro-crystals.

Here, plots represent the evolution over time for the weight of the protein droplet, together with the calculated protein concentration and precipitant concentration. Shown here is a recorded photograph of Thaumatin micro-crystals at the end of the experiment. The droplet shows a very abundant amount of small micro-crystals saturated in protein solution.

Once mastered, this technique can be done in one hour and 30 minutes, if it's done properly. While attempting this procedure, it is important that you adjust the DLS laser in the microdosage systems into the protein droplet. After watching this video, you should have a good understanding how to easily handle this crystallization device in order to successfully crystallize a protein sample.

This method is possible due to the in situ dynamic light scattering which is directly coupled to the crystallization droplet. This allows us to monitor all changes in the radius distribution and therefore, we can pinpoint certain distribution patterns to either crystallization or protein precipitation. Following this procedure, other methods like cryo-electron microscopy or electron diffraction can be applied to answer additional questions and to verify the crystalline state of sub-micrometer sized particles in solution.

The implications of this technique extend towards the two-step theory of nucleation, as the DLS maps are showing a clear pattern of a double-sized distribution of particles in solution, which are prior to crystal growth.