Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

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This method can help answer key questions in the biochemistry field such as size, shape and conformational changes of biomolecules and their complexes. The main advantage of this technique is that experiments can be performed under physiologically relevant buffer conditions in an environment where biomolecules are stable. SAXS is a complimentary technique to many other biophysical and structural biology methods.

Combining SAXS with these methods can help us in developing structure-based therapeutics. Though this method can provide insight into biomolecular complexes, it can also be applied to other systems such as studying nanoparticals and drug delivery versicles. Generally, individuals new to this method will struggle because it appears as an overwhelming and complicated process, to go from raw scattering data to low resolution structures.

Visual demonstration of this method is critical as the pipeline of data analysis to obtain structural models from raw data is difficult to learn because it involves the use of many different packages of software. There are a few software packages that are useful for SAXS data analysis. These include SCATTER, BioXTAS RAW and the ATSAS suite.

In this video, we will provide an overview of the general steps to be taken when analyzing raw SAXS data, using the ATSAS program suite. To begin, install and load the ATSAS program suite. Open the PRIMUS/Qt program and go to the Open Menu option.

Here, select up to 13 data files of interest. The data files must be in the ASCII format, in which the first column is the s-vector axis and the second column is the intensity. Repeat this step for the buffer only data, inserting this data into a second Tools menu.

Next, navigate to the Data Processing window and select Subtract. This will generate a subtracted scattering curve, representing just the scattering from the macromolecule of interest. To perform Guinier analysis, start with a buffer subtracted scatter curve loaded into PRIMUS/Qt.

Next click on Radius of Gyration, which will open the Primus Guinier Wizard. At this point, a plot showing the natural log of the intensity versus scattering angles squared will be displayed. To obtain a preliminary radius of gyration, find the Command Prompt window and use the Autorg function.

After pushing Autorg, click the upper limit up one, then down one to force the program to update the statistical measurements. Alternatively, input multiple files at once by clicking the same open prompt and selecting multiple data files names by highlighting them in the same manner as previously described. To begin Kratky analysis, click to select the data file name.

This will plot the data in the window. And then, select Plot. Next, below the Plot button, click on Kratky Plot.

As the result, globular proteins display a GALC-ean peak while unfolded proteins will display a plateau instead of a peak and resemble a hyperbolic plot like the one shown here. Load buffer subtracted data for each concentration in PRIMUS/Qt once again. And under the Processing tab, click Scale and inspect each curve and the i-scale number, which correlates to the dilutions made from the original sample.

Following inspection, merge the data by clicking on the Merge button in the Processing window. To generate the distance distribution plot, load the merged data curves into PRIMUS/Qt and then click Distance Distribution in the Analysis tab. Adjust the data range of the merged data to avoid any significant noise at the tail end of the raw data.

Also, omit data points close to the beamstop in the low-q region by selecting the lower range value and increasing the number. To determine the Dmax, start with a range of five times the radius of gyration obtained from the Guinier analysis. Then, gradually decrease this value until the distance distribution plot does not abruptly drop to zero on the y-axis and does not have a long tail before approaching zero.

Check that the experimental radius of gyration versus intensity plot, which is derived from the Guinier approximation and the distance distribution radius of gyration versus intensity numbers are similar. Here, the intensity of scattered light is shown plotted against scattering angle, suggesting both the quality of the biomolecules nidogen-1, laminin gamma-1 and their complex, as well as their shape. The electron pair distance distribution determined from the scattering data suggests that the biomolecules have an elongated shape and the Kratky plot suggests that the proteins are in a folded state, as the data tends to plateau, or even increase in the larger q-range and lack a bell curve shape.

In addition the Guinier plots, which uses data at low scattering angle, indicates the linear region for determination of the radius of gyration, which is shown here for nidogen-1, laminin gamma-1 and their complex. To study protein-protein complexes, MONSA was used to obtain the low resolution structure of the entire nidogen-1 laminin gamma-1 complex, suggesting that only the C-terminal region of both proteins participate in mediating interactions whereas the rest of the domains are far apart from each other. Don't forget that working with Synchrotron radiation can be extremely hazardous and precautions and safety outlined by each different beamline should always be taken while performing initial data collection.

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Journal (Biochemistry)

Journal (Biochemistry)

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

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