Solving the structure of the ryanodine receptor domain can help with our understanding of the molecular mechanism of the protein function, insecticide action, and insecticide resistance development. This measure implies the use of X-ray crystallography for structure illustration, which is considered a gold standard for protein structure determination at near-atomic resolution. This high-resolution structure of the insect ryanodine receptor domain reveals the mechanism of channel gating and provides an important template for the development of species-specific insecticides using structure-based drug design approaches.
Generally, individuals new to this method will struggle, because prospective protein crystallographers must be proficient in biochemistry, biophysics, computer science, and maths. Begin by amplifying the DNA corresponding to the protein of interest by polymerase chain reaction. At the end of the reaction, run the entire 50 microliters of reaction mix on a 2%agarose gel, and extract the DNA with a gel extraction kit according to standard protocols.
Next, linearize the LIC vector DNA by mixing 20 microliters of the vector DNA with 6 microliters of 10X reaction buffer and 4 microliters of SspI restriction enzyme in 30 microliters of double-distilled water. Incubate this reaction mixture for three hours at 37 degrees Celsius. At the end of the incubation, run the entire reaction mix on a 1%agarose gel, followed by extraction of the linearized vector DNA with a gel extraction kit according to the manufacturer's instructions.
Next, perform separate T4 DNA polymerase treatments on 5 microliters of the linearized vector DNA and the PCR amplified insert DNA, according to standard protocols. Incubate for 40 minutes at room temperature, followed by 20 minutes of enzyme heat inactivation at 75 degrees Celsius. Perform the ligation-independent cloning annealing reaction by combining 2 microliters of the T4 treated insert DNA with 2 microliters of the T4 treated LIC vector DNA for a 10 minute incubation at room temperature.
To transform BL21(DE3)E. Coli cells with the recombinant plasmid, thaw 50 microliters of competent cells on ice, and add approximately 1 microliter of the annealed plasmid to the tube. Gently flick the tube two to three times to mix the cells and the DNA, and place the tube back on ice for 20 minutes.
At the end of the incubation, heat-shock the cells at 42 degrees Celsius for 40 seconds, followed by two minutes back on ice. Next, add one milliliter of room-temperature LB medium to the tube, and place the cells on a 250 RPM shaker for 45 minutes at 37 degrees Celsius. At the end of the shaking incubation, plate 150 to 200 microliters of this mixture onto selection plates, and invert the plates overnight at 37 degrees Celsius.
The next day, culture a single colony in 100 milliliters of 2-YT medium, supplemented with kanamycin, in a shaker incubator at 37 degrees Celsius overnight. The next morning, inoculate one liter of 2-YT medium, supplemented with kanamycin, with 10 milliliters of overnight culture, and incubate at 37 degrees Celsius with shaking until the OD600 reading reaches approximately 0.6. Then, induce the culture with IPTG to a final concentration of 0.4 millimolar, and grow the cells for five hours at 30 degrees Celsius.
At the end of the incubation, harvest the cells by centrifugation, and re-suspend the pellet to a 10 grams of bacteria per 40 milliliters of lysis buffer concentration. Disrupt the cell walls by sonication at a 65%amplitude, and one second on one second off, for eight minutes. Remove the cell debris by centrifugation.
Then, filter the supernatant though a 22 micrometer filter, and load the solution into a sample loop. To purify the fusion protein, inject the filtered supernatant from the sample loop into a five milliliter nickel-nitrolotriacetic column on a purification system with a linear gradient of 20 to 250 millimolar imidazole. Cleave the eluted target protein with tobacco etch virus protease, at a 1-to-50 ratio, overnight at 4 degrees Celsius, and purify the cleavage reaction mixture on an amylose resin column, and a nickel-nitrolotriacetic column, to remove the tag and the tobacco etch virus protease.
Dialyze the flow-through from the nickel-nitrolotriacetic column against dialysis buffer to reduce the salt concentration, and purify the sample on an anion exchange column by a linear gradient of 20 to 500 millimolar potassium chloride in the elution buffer. Then, concentrate the protein in a centrifugal concentrator. Inject the concentrated protein into a Superdex 200 26/600 gel filtration column to check the homogeneity, and assess the purity of the protein by 15%SDS-PAGE.
To prepare the protein for crystallization, concentrate the purified protein sample to 10 milligrams per milliliter in the centrifugal concentrator, and buffer exchange to crystallization buffer before 80 degree Celsius storage. To perform crystallization screening, use the sitting drop vapor diffusion method at 295 Kelvin, with several crystallization kits, and an automated liquid-handling system in a 96-well format. At the end of the drop setting, seal the 96-well crystallization plate to prevent evaporation, and to enable the equilibration of the protein drop within the reservoir buffer.
Place the plates in a crystal incubator at 18 degrees Celsius. Check the plates periodically under a light microscope to monitor the crystal formation and growth. To differentiate the protein crystals from the salt crystals, add one microliter of protein crystal dye to the target drop, and observe the crystals under the microscope after one hour.
The protein crystals will appear blue. To further optimize the crystals, use the positive crystallization conditions and the hanging drop vapor diffusion method in 24-well plates, followed by incubation in the crystal incubator at 18 degrees Celsius. To determine the protein structure, mount the crystals on a cryoloop under a light microscope for flash cooling in liquid nitrogen, and place the crystals in a unibuck for storage and transportation.
Pre-screen the crystals in an in-house X-ray diffractor, using the manual centering function to mount and center the crystals. Then, use the X-ray diffraction software to collect the data. To index, integrate and scale the data set, using HKL-2000 suite, first, select the detector, and load the data set.
Then, carry out the peak search function to find the diffraction spots. Index the spots, select the right space group, and perform peak integration. Next, scale the data set.
Adjust the error model and scale again. Save the output sca file. To determine the structure using Phenix software suite, create a new project.
First, run Extriage with the sca file to calculate the possible copy number of protein molecules in the asymmetric unit. Next, to solve the phase problem by molecular replacement, run Phaser, using the diffraction data file, the template structure file with high-sequence identity and structural similarity as the target protein, and the protein sequence file, to find the solution. Perform auto-build in Phenix to generate the initial model, using the output file from Phaser and the sequence file from the target protein.
Manually build the structure into the modified experimental electron density using COOT, and refine using Phenix refine in iterative cycles. Validate the final model using the validation tools in Phenix. In this representative experiment, purification of the end-terminal domain of the diamondback moth ryanodine receptor protein, as demonstrated, yielded a single band at about 21 kilodaltons by STS-PAGE analysis.
The elution volume from the gel filtration column confirmed the purified ryanodine receptor end-terminal domain to be a monomer. For crystallization, the most optimal conditions under which high-quality plate-shaped crystals were formed was in the presence of 1 molar HEPES of a pH of 7, and 1.6 molar ammonium sulfate. Determination of the protein structure from the diffraction data set utilizing softwares as demonstrated revealed the diamondback moth ryanodine receptor end-terminal domain covering residues 1 through 205.
Proteins with large disorder, flexible regions, or with weak affinities are challenging to crystallize. In these scenarios, protein engineering strategies such as surface entropy reduction, loop truncation, and cross-linking, may increase the likelihood of obtaining better protein crystals. In addition to revealing high-resolution protein structures, X-ray crystallography may also be used to study protein-pesticide interactions, which could help with structure-based pesticide design.
