Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

Summary

The demonstration of the small and wide angle X-ray scattering (SWAXS) procedure has become instrumental in the study of biological macromolecules. Through the use of the instrumentation and procedures of specific angle methods and preparation, the experimental data from the SWAXS displays the atomic and nano-scale characterization of macromolecules.

Abstract

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique where X-rays are elastically scattered by an inhomogeneous sample in the nm-range at small angles (typically 0.1 - 5°) and wide angles (typically > 5°). This technique provides information about the shape, size, and distribution of macromolecules, characteristic distances of partially ordered materials, pore sizes, and surface-to-volume ratio. Small Angle X-ray Scattering (SAXS) is capable of delivering structural information of macromolecules between 1 and 200 nm, whereas Wide Angle X-ray Scattering (WAXS) can resolve even smaller Bragg spacing of samples between 0.33 nm and 0.49 nm based on the specific system setup and detector. The spacing is determined from Bragg's law and is dependent on the wavelength and incident angle.

In a SWAXS experiment, the materials can be solid or liquid and may contain solid, liquid or gaseous domains (so-called particles) of the same or another material in any combination. SWAXS applications are very broad and include colloids of all types: metals, composites, cement, oil, polymers, plastics, proteins, foods, and pharmaceuticals. For solid samples, the thickness is limited to approximately 5 mm.

Usage of a lab-based SWAXS instrument is detailed in this paper. With the available software (e.g., GNOM-ATSAS 2.3 package by D. Svergun EMBL-Hamburg and EasySWAXS software) for the SWAXS system, an experiment can be conducted to determine certain parameters of interest for the given sample. One example of a biological macromolecule experiment is the analysis of 2 wt% lysozyme in a water-based aqueous buffer which can be chosen and prepared through numerous methods. The preparation of the sample follows the guidelines below in the Preparation of the Sample section. Through SWAXS experimentation, important structural parameters of lysozyme, e.g. the radius of gyration, can be analyzed.

Protocol

1. Preparation of the Sample

1. Use a needle to remove some of the sample from the sample container.*
2. Use the needle to fill the capillary (maximum diameter of 2.2 mm) with sample. The capillary must be filled between 2 and 3 cm from the bottom.
3. Close the capillary by melting wax on its tip.
4. Unscrew the vacuum sample holder from the system.
5. Take the capillary by the fused end (wax end) and insert the un-fused end into the sample holder.
6. Place the holder back into the system and screw in place.

* Solid samples (including powder samples) can be directly placed in the sample hol....

Results

SAXS and WAXS altogether can provide structural information of the sample through the following parameters: the radius of gyration, particle size and shape, solution structure factor, specific inner surface and pore size, lattice type and dimension, and electron density. SAXS and WAXS can also be applied to the study of protein dynamics ¹.

The structural information of SWAXS experiments is obtained by comparing the experimentally detected spectra and the computational results of the.......

Discussion

The comparative procedure of the SWAXS system allows for numerous variables to be determined from experimental analysis. The parameters that are attained from the analysis can be used for different purposes according to the sample and experimental setup. SAXS provides information about nano-scale size and shape of the object, whereas WAXS focuses on the atomic and micro-scale structure (e.g. molecular lattice, unit cell dimension symmetry). More specifically, for particles in dilute solutions, SAXS can st.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
The System3 Small- and Wide-Angle X-Ray Scattering (SWAXS) Camera	Hecus XRS and IBN, Graz, Austria
GNOM	ATSAS 2.3 package by D. Svergun EMBL-Hamburg