The overall goal of this procedure is to produce a recombinant protein that is composed of a mixture of ordered and disordered domains for hybrid structural analysis by both x-ray crystallography and small angle x-ray scattering. This method can help to answer key questions in structural biology about putting biological function so that the determination of all solutions stay structure of protein. The main advantage of this technique is that it can be used to model dynamic regions of a protein or macro-molecule that cannot be resolved by x-ray crystallography.
Generally, individuals new to this method will struggle at the beginning with learning how to properly assess the quality of their SAXS data. To begin the procedure, transform an Nsa1 expression plasmid into a suitable E.coli expression strain. Culture the cells, induce Nsa1 expression, and harvest the cells.
Resuspend the cells in 25 milliliters of lysis buffer, pre-chill to four degrees Celsius, containing one EDTA-free protease inhibitor tablet. Sonicate the cells at four degrees Celsius for seven minutes, with a cycle of two seconds on, two seconds off. Then, centrifuge the lysate at 26, 900 times gravity for 45 minutes at four degrees Celsius.
Ensure that the mixture does not freeze during centrifugation. Next, equilibrate a gravity flow column containing 10ml of immobilized cobalt affinity resin with lysis buffer. Load the clarified lysate onto the column and allow it to flow through the resin at four degrees Celsius.
Then, wash the resin twice with 100ml portions of lysis buffer. Elute Nsa1 from the column with 20ml of elution buffer. Run a 15mcl aliquot of the eluere on a four to 15%SDS-PAGE gel to confirm the elution of the protein.
Then, add 1ml of the 1mg/ml TEV protease stock solution to the eluere. Incubate the eluere at four degrees Celsius overnight to remove the MBP tag. Then, use a 10 kilodalton centrifugal filter unit to concentrate the MBP-cleaved Nsa1 to about 5ml, being careful not to over-concentrate.
Purify the concentrated protein on a gel filtration column pre-equilibrated with Buffer A.One of the critical steps in this procedure is ensuring you remove all aggregates during the purification. Aggregates will interfere with crystallization and SAXS data acquisition and analyses. Analyze 15mcl samples from each column fraction with SDS-PAGE to confirm the separation of MBP from Nsa1.
Combine the Nsa1 containing fractions. Use a 10 kilodalton centrifugal filter unit to concentrate the combined fractions to about 8mg/ml. Measure the absorbance at 289 nanometers and calculate the protein concentration.
Immediately proceed to proteolytic screening and crystallization trials. To begin the proteolytic screening, prepare 1mg/ml stock solutions of Alpha-comotrypsin, trypsin, elastase, capayin, septilisin, and Endoproteinase GluC. Dilute each stock solution with dilution buffer to 1:10, 1:100, and 1:1, 000.
Dilute the 8mg/ml solution of Nsa1 to 1mg/ml with Buffer A.Then, for each protease dilution, combine 1mcl of the protease dilution with 9mcl of the diluted protein stock. Incubate the solutions at 37 degrees Celsius for one hour. Then, add 10mcl of 2x SDS-PAGE sample to buffer to each solution and heat the solutions at 95 degrees Celsius for five minutes.
Run the solutions on a four to 15%SDS-PAGE gel to identify protease resistant domains. Create truncated expression constructs that exclude protease labile regions. Express and purify these proteins for crystallization optimization.
After acquiring SAXS data for the Nsa1 protein over a range of concentrations, set the terminal shell file path to the directory containing the data. Then, launch the analysis software. Load the scattering curves into the analysis software.
Use the auto RG tool to determine the radius of gyration and the forward-scattering intensity for each curve. Then, generate Kratky plots for each scattering curve to determine the degree of compactness. Use the autonome tool to calculate the pair-wise distribution function for each curve, using a starting maximum dimension of 3x the radius of gyration.
Optimize the maximum dimension to obtain a smooth, pair-wise distribution function curve that is consistent with the experimental scattering curve. Compare the structural parameters across concentrations to verify the absence of radiation damage or concentration-dependent effects. It is critical to compare key structural parameters, including radius of gyration, molecular mass, forward scattering and pair distance distribution functions across a concentration series.
And that's to ensure there's no radiation damage or concentration effects. Next, run the ensemble optimization method utility using the starting protein crystal structure file, the target protein sequence file, and the experimental SAXS data file. Compare the chi squared values from the starting crystal structure file to the ensemble file to determine whether the ensemble model fits the experimental data well.
Use molecular visualization software to overlay the crystal structure with the generated conformers. Note the total number of conformers and the fraction of occupancy for each conformer contributing to the scattering curve. Proteolytic cleavage of the protease labile C terminus of Nsa1 resulted in the formation of orthorhombic crystals.
The freshly purified Nsa1 formed cubic crystals, but the crystal structure still lacked electron density for the labile c terminus. The n-terminal seven blated beta propeller WD-40 domain of Nsa1 was well-resolved in both structures. The Nsa1 WD-40 domain alone was not a good fit for the experimental SAXS data.
A combination of rigid-body modeling and abinitial reconstruction of the C terminus significantly improved the goodness of fit. Ensemble modeling provided an even greater improvement in goodness of fit, revealing the confirmational sampling of the C-terminal tail in solution. While SAXS is a low-resolution structural technique, it compliments very well high-resolution structural studies, like x-ray crystallography.
So you can model flexible and disordered regions. By collecting and analyzing SAXS data of macromolecule, you can begin to understand confirmational change that occurred upon After watching this video, you should have a good understanding of how to solve the structure of a protein composed of ordered and disordered regions by both x-ray crystallography and SAXS.
