Small angle X-ray scattering (SAXS) from biological macromolecules such as proteins (also called bioSAXS) gives information on the size and shape of macromolecules in solution and can provide details of the radius of gyration and molecular weight of a protein complex (e.g. multi sub-unit complexes), significant conformational changes upon ligand binding, screening of structural changes with formulation conditions (e.g. pH, ionic strength, additives, temperature etc) and many more applications.
The high intensity X-rays available at a synchrotron provide significant benefits over lab-based instruments.
Firstly the high intensity of the X-rays at a synchrotron means that measurement times are much faster, allowing structural investigation of macromolecule solutions under relevant conditions, for example at different temperatures. SAXS at Diamond can provide reliable access to second or shorter time scales, essential for understanding protein solution structures and to minimse the impact of radiation damage. Information can be obtained on the shape and size of proteins and macromolecules which may not be possible using other techniques. Small beam sizes mean that only small volumes (35µL) are required for measurements.
Shorter measurement times also result in increased sample measurement and high throughput is a key advantage of synchrotron bioSAXS measurements. A BIOSAXS robot available on B21 uses 96 well plates which can be chilled until needed for measurements ensuring high quality samples. The shorter measurement times also allow SAXS to be performed in conjunction with other techniques (for example size exclusion chromatography, SEC) called SEC-SAXS where online protein purification ensures high purity samples and ideal buffer matching.
Secondly, the greater X-ray intensity allows bioSAXS measurements to be performed at a greater separation between the sample and the detector, which allows access to a much wider range of length scales (particularly for larger length scales) than achievable using a lab instrument.
There are a vide variety of applications for macromolecular SAXS experiments but popular bioSAXS applications include:
Diamond has a dedicated solution state Small Angle X-ray Scattering (SAXS) beamline that can accommodate a wide variety of aqueous-based and some limited organic solvent-based samples. In order to achieve high sample through put, B21 is equipped with a highly automated BIOSAXS robot for small volume liquid handling from a 96 well plate.
B21 also offers Size-Exclusion Chromatography (SEC) coupled SAXS with either Superdex or Shodex SEC columns controlled by an Agilent HPLC. SAXS samples are measured through a temperature controlled capillary. The beamline users benefit from a fast streamlined data processing and data analysis pipeline so data can be analysed during the experiment.
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