The overall goal of this procedure is to produce protein crystals for X-ray diffraction experiments here. Three different techniques for crystal screening are demonstrated vapor diffusion crystallization methods including hanging drop and sitting drop, as well as micro batch crystallization methods are described for vapor diffusion crystallization. The wells are filled with precipitant.
Then a high concentration of purified protein sample is mixed with the precipitating agent. The mixed drop is then sealed in a well containing the precipitant solution. Micro batch crystallization begins with filling the well with paraffin oil.
The protein is then loaded into the wells, followed by the precipitant solution. The final step of all the methods is to let the system equilibrate and to follow crystal appearance and growth within the drops. Ultimately, crystals can be obtained from this technique and the protein diffraction pattern can be determined using an x-ray source with a suitable detector.
Hi, I'm Sal from the laboratory of Professor Yoga Motis in the Department of Molecular biophysics and biochemistry at Yale University. Today we'll show you a procedure for the crystallization of a purified protein. We use this procedure in our lab to study the structural basis of viral infection.
So let's get started and grow some crystals. To identify a crystallization method for a macromolecule of choice, initial screens can be performed using commercially available crystallization solutions that are chosen to exploit the sparse matrix incomplete factorial method of trial conditions. These conditions are selected based on known and commonly successful crystallization conditions for macromolecules.
Once the crystallization condition is known, a narrow screen can be assembled varying the precipitant concentration in pH around the original hit. Usually the crystallization conditions used for these screens consist of a precipitating agent assault and a buffer to set the pH, all stock solutions must be filtered through a 0.22 micron filter for this demonstration. The previously identified crystallization conditions for chicken lysosome are used with sodium chloride as the precipitant begin the procedure by preparing one milliliter solutions of various concentrations of sodium chloride in 0.1 molar sodium acetate of various phs.
Next thaw the protein sample is stored at minus 80 degrees Celsius and keep it on ice. Typical protein concentrations are in the range of five to 50 milligrams per milliliter with higher concentrations, usually giving better results. Here, a 50 milligram per milliliter solution of hen egg lysosome in 0.02 molar sodium acetate pH 4.9 is used.
Spin the sample for 15 minutes at 18, 000 G and four degrees Celsius to remove any partially aggregated proteins or precipitate from the thawing step. These aggregates can interfere with crystal growth and promote further aggregation. Following centrifugation.
Determine the protein concentration from its absorbance at 280 nanometers using a UV spectra photometer. The protein concentration can be calculated from the absorbance reading and the protein's extinction coefficient. Once the protein sample is ready, fill a 24 well hanging sitting drop crystallization tray with 500 microliters of precipitant solution according to the tray map.
Create a silicone grease ring around the edge of each well without perfectly closing the ring so that air can escape upon well.Ceiling. Clean the cover slide with condensed air spray or a professional wipe to avoid dust, which will ease the interpretation of the crystallization drops and eliminate contaminations. The next step is to pipette equal volumes of the protein sample and reservoir solution onto the cover slide.
However, different volumes of protein and precipitant may be tried as part of the optimization process using more protein than precipitant results in a net concentration of the protein in the equilibrated drop, which may be desirable for dute protein samples or to slow crystal nucleation or growth. For this demonstration, two microliters of 50 milligrams per milliliter lysozyme in 0.02 molar sodium acetate. pH four is loaded in the center of the cover slide, followed by two microliters of crystallization solution.
When pipetting protein and reservoir solution onto the cover slide, be extremely careful to avoid bubble formation, which often occurs when air is blown out of the pipette. Gently mix the drop to help prevent premature protein precipitation due to locally high precipitant concentrations in the drop mixing is particularly important for viscous precipitants such as polyethylene glycol using tweezers. Flip the cover slide gently and lay it down on the grease ring on top of the, well perform this in a directional manner so that air can escape the well.
Otherwise, the air can raise the cover slide compromising the well seal. Move on to the next well until the tray is complete. Once the tray has been set up, visually inspect it, eliminating dust particles and other contaminants which can reduce the false positive identification of protein crystals.
Use a score sheet to document the experiment. Next, place the tray at the desired incubation temperature generally between four degrees Celsius and room temperature. The most successful temperature is 20 degrees Celsius.
Take care to handle the tray gently and avoid any shaking vibrations or temperature changes during transfer or incubation can prevent crystal growth or negatively affect crystal quality. Check the tray for crystals the following day and then every few days, always handling the trays with care. Document any findings and drop morphologies in a tray Specific scoring sheet.
Crystals typically take two to five days to appear, although crystals occasionally appear almost immediately or up to several months later. Once crystallization conditions have been identified and the growth rate of the crystals is known, it's best to leave the trays undisturbed until crystals have appeared and have grown to at least half of their final size. Leaving the trays undisturbed can reduce the number of crystal nucleation events, thus resulting in smaller numbers of larger crystals for sitting drop crystallization follow the same procedure as for hanging drop, except that we'll use a 24 well sitting drop tray in which cover slides are not required.
Instead, the well contains a shelf on which the protein and precipitant are mixed pipette, two microliters of 50 milligrams per milliliter lysozyme solution onto the center of the shelf, avoiding the generation of air bubbles. Next, add two microliters of precipitant solution to the sample after tray setup. Seal the wells with optical tape with sitting drop.
No cover slides are necessary. Continue the incubation and crystal scoring as described for hanging drop. To perform a micro batch procedure.
Prepare the crystallization solution and protein as described for the hanging drop procedure. Obtain a new micro batch tray and air spray to avoid dust and other large particles in the micro batch tray. Add paraffin oil to each well to a depth of three millimeters.
Then remove excess oil. Next load one microliter of the protein solution into the prefilled well, making sure the drop sinks to the bottom of the well. Then load one microliter of precipitant solution into the well.
Be sure the drop sinks to the bottom of the well and fuses with the protein droplet. Move to the next well until the tray is complete and continue with the incubation and crystal analysis as described here. Typical outcomes of protein crystallization.
Experiments are shown. Amorphous precipitation occurs when the protein and or precipitant are in high concentration. Conversely, drops that are unsaturated usually remain clear.
Phase separation can occur if the protein or detergent separates to a different phase when mixed with certain precipitants at high concentration. Under optimal conditions, protein crystals should form Arabidopsis csn. Seven protein crystals that were obtained in polyethylene glycol 8, 000 and magnesium acetate appear as rod-shaped.
Crystals also shown are lysosome protein crystals that were obtained in one molar, sodium chloride and sodium acetate. pH 4.9. This time-lapse movie demonstrates that lysosome crystals grow into large prisms within a few hours of setup when using the hanging drop method.
We've just shown you how to set up a protein experiment when you're doing this procedure. It is important to work in an environment that's steady control for temperature and humidity as high humidity helps to minimize drop evaporation, try to adopt a technique that minimizes exposure of the drop to the open air. For this reason, it's essential to operate in a well organized work environment.
So that's it. Thanks for watching and good luck with your experiments.
