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The overall goal of this procedure is to efficiently grow single lattice diffraction quality crystals of recombinant proline proline endopeptidase-1, or rPPEP-1, protein. Without this method, rPPEP-1 instantly produces highly intergrown crystals not suitable for xray diffraction analysis. The main advantage of this technique is that a high number of well ordered and well diffracting crystals for structural determination of rPPEP-1 can be produced in a short period of time and with limited material input.

This procedure makes use of the microcity method and can be adapted for every successful initial crystallization condition of rPPEP-1 as well as its substrate peptide complexes. This method can be adapted to produce well ordered crystals of virtually every protein that yields intergrown crystals. It can also be used in cross seeding experiments, protein variance, or in cocrystallization experiments with small molecules.

Visual demonstration of the handling of crystallization methods is crucial, since even small variations can have a significant impact on the diffraction quality of the crystals. Carry out crystallization trials in the sitting drop format using standard commercially available screens and a crystallization robot. First, concentrate the purified protein to 12 milligrams per milliliter using a centrifugal ultrafiltration device in five minute intervals at 4000 times g and four degrees Celsius.

Mix the protein after each interval to prevent precipitation and aggravation. Determine the protein concentration at 280 nanometers by using the extinction coefficient of 25, 900 per molar centimeter. After equilibrating the protein to 20 degrees celsius clear away all particles and dust by centrifugation for 10 minutes at 16000 times g and 20 degrees celsius.

To perform crystal screens, use prefilled crystallization plates sealed and stored at four degrees celsius. When setting up trials, work swiftly, as the small volumes quickly dry out. Also use a humidity chamber around the dock of the robot if possible.

After equilibrating all crystallization plates to 20 degrees celsius, set up the screen by pipetting protein ent reservoir into subwells two to four. Use a drop volume of 300 nanoliters. Use protein to reservoir ratios of 200:100 for subwell two, 150:150 for subwell three, and 100:200 for subwell four.

Immediately seal the plate and place it in a chamber at 20 degrees celsius. Inspect the trays after setup every day during the first week and then follow with weekly inspection. For a cocrystallization of the substrate peptide rPPEP-1 complexes, mix rPPEP-1 at 24 milligrams per milliliter in a 1:1 ratio with a sevenfold molar excess of peptide solution lay off lyophilized powder solubilized in tris buffered saline.

After incubating for 30 minutes at 20 degrees celsius, clear away all particles and dust by centrifugation for 10 minutes at 16000 times g and 20 degrees celsius. Proceed with crystallization using the same microseeding procedure as for the unbound rPPEP-1 protein. Highly intergrown crystals of rPPEP-1 appeared after two days in a condition containing 2.4 molar ammonium phosphate dibasic and 0.1 molar trisapseal pH 8.5.

To optimize the initial condition, use software to calculate the volumes and the pipetting scheme to obtain two milliliters of each condition that allows for 10 optimization screens. Conditions include 1.8 to 2.55 molar ammonium phosphate dibasic in steps of 0.15 molar as well as 0.1 molar trisapseal pH 7.5 to 9.0 in steps of 0.5 pH units. Prepare a grid screen comprising 24 conditions from stock solutions.

Use the pipetting scheme to compose the individual conditions. Pipette 200 microliters of every grid screen solution into the wells of a 24 well plate and equilibrate the plate at 20 degrees celsius. Manually set up the crystallization plate.

Use a drop volume of three microliters and a protein to reservoir ratio of 2:1, 1.5:1:5, and 1:2. Here, use a positive displacement pipette to avoid air bubble formation. Immediately seal the plate.

Then place the plate in a chamber at 20 degrees celsius. After one to four days, highly intergrown crystals of rPPEP-1 appear in the four conditions containing 2.55 molar ammonium phosphate dibasic and 0.1 molar trisapseal pH 7.5 to 9.0, whereas no crystals are formed in the remaining 20 conditions. Perform the microseeding procedure to obtain single crystals of rPPEP-1 in these conditions.

To prepare a microseed stock, first choose a single intergrown crystal from one of the successful conditions. Next, transfer 50 microliters of the respective mother liquor into a 1.5 milliliter tube containing a small, highly polished glass bead and one microliter of the mother liquor to the glass cover slide. Using a mounted nylon lube, fish out the crystal and transfer it into the drop on the glass cover slide.

Then transfer the liquid containing the crystal into the tube and vortex at high speed for 30 seconds, making sure the glass bead is swirling. Make a 1:1000 dilution of the seed stock into a new 1.5 milliliter tube containing the same freshly prepared condition and vortex thoroughly for five seconds. Next, remove the seal of the plate covering the 20 conditions with clear drops.

Pipette 0.5 microliters of the seed stock into the wells. Seal the plate and place it into a chamber at 20 degrees celsius. After applying this microseeding technique, single crystals of high diffraction quality appear several hours to several days after setup in various conditions, containing 1.8 to 2.4 molar ammonium phosphate and 0.1 molar tris pH 7.5 to nine.

Crystals grow to a size of 100 to 200 microns in the largest dimension. Choose the optimal size of nylon loop for the maximal length of the chosen crystals by placing the loop on the sealing tape just above the crystal and focusing up and down. The typical longest access of rPPEP-1 crystals is about 100 to 200 microns.

Also prepare the appropriate cryocondition. Fill the foam dewers with liquid nitrogen. Then load the vial clamp with a vial and precool it in the liquid nitrogen filled 800 milliliter foam dewer.

Place a cryocane holder marked with a suitable identifier and a cryosleeve in the liquid nitrogen filled two liter foam dewer. Then load the magnetic wand with the mounted nylon loop of the right size. Next, cut open the sealing tape on the crystallization plate with a sharp scalpel and remove it with forceps.

Pipette one microliter of the cryocondition onto the cover slide. It is particularly important to work fast at this step, as drying out the crystal or the tiny volume of cryosolution in the loop could lead to fluctuations in crystal quality or even a complete loss of diffraction. All crystal manipulation steps should be performed under the stereo microscope.

If the crystal sticks to the plastic surface, detach it from the ground by deforming the surrounding plastic with an acupuncture needle. Remove the crystal from the drop by fishing it out with the mounted nylon loop. Quickly transfer the crystal to the drop of cryocondition and let it equilibrate for one second.

Then fish the crystal out of the drop with the mounted nylon loop as quickly as possible. Immediately plunge freeze the retrieved crystal in liquid nitrogen. When the liquid nitrogen around the mounted loop stops boiling, place the loop in the vial.

Place the vial on the cryocane holder. Once the holder is loaded with six vials, place a cryosleeve around the holder. Store the crystals in a tank filled with liquid nitrogen until use.

To perform data collection, carefully retrieve the mounted crystal from the cryocane using a clamp and store it in a foam dewer for transport. Move the beam stop and the detector into the park position and move the cryonozzle up by some millimeters. Mount the base of the cryocap onto the goniometer head by retaining the base with one's fingers while quickly removing the vial.

Then move the cryonozzle and beam stop back into place and center the crystal. At all times, be careful not to touch the beam stop or detector. Proceed to collect the 180 degree data set with an oscillation angle of 0.1 degrees, as shown here.

Shown here are highly intergrown rPPEP-1 crystals obtained from initial screening using commercial crystallization screens in 1.4 sodium citrate tribasic dihydrate, 0.1 molar HEPES sodium, pH 7.5. Shown here are crystals obtained from initial screening with 60%volume to volume tacsimate, pH 7.0, 0.1 molar BIS-TRIS propane, pH 7.0. Crystals also appeared in 2.4 molar ammonium phosphate dibasic, 0.1 molar tris, pH 8.5.

After applying the microseeding optimization procedure, single crystals were contained in several conditions, including 2.1 molar ammonium phosphate dibasic, 0.1 molar tris, pH 8, and 2.25 molar ammonium phosphate dibasic, 0.1 molar tris, pH 8. The crystals mounted in nylon loops are of 100 to 200 microns in size and appear to have a single lattice from microscopic inspection. Xray diffraction analysis shows that these crystals indeed exhibit a single lattice and diffract xrays to near atomic resolution.

Solving the crystal structure of the recombinant PPEP-1 substrate peptide complex gives insight into the substrate binding mode of PPEP-1. The substrate peptide could diffuse into the substrate binding group of PPEP-1 in its open confirmation. Then as loop closure would occur, the substrate interacts with PPEP-1 via hydrogen bonds and the unique aliphatic-aromatic side chain network located on the S-loop.

The substrate binds in a unique double kinked conformation with kinks at the sizzle peptide bond and the P2 prime to P3 prime peptide bond. After watching this video, you should have a good understanding of how to produce single lattice well diffracting crystals of recombinant PPEP-1 for structural studies. Similar approaches can be used with other proteins, yielding highly intergrown crystals and further varied incubation temperature and C-stock dilution.

Due to its versatility and simple use, this simple technique should be tried in every crystal optimization process.