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Industrial Liaison Group:
Tel: +44 (0) 1235 778797
E-mail: industry@diamond.ac.uk
Solution small angle X-ray scattering (also known as protein SAXS or bioSAXS is a powerful tool for analysis of the structure of proteins in solution. Although care must be taken to ensure that the samples are pure and free from aggregation in advance of the measurement (as this is assumed for data analysis and cannot be measured directly), it can be used for a wide range of protein types and solution conditions.
The resolution of SAXS is about 15 Å.
B21 is now available for industrial users and we offer a full range of bioSAXS services including a full data collection and data analysis service.
Please contact us for the latest availability and for any further information.
Key advantages of protein SAXS include:
• Determination of protein molecular weight. Determination of the molecular weight of a protein or protein complex and comparison to the theoretical values can give information about the oligomerisation state of the sample.
• Structure validation. X-ray crystallography is the dominant method of structure determination, however because of the nature of crystal packing, the solution structure or oligomeric state may not be clear and may be ascertained using bioSAXS. Protein SAXS has also been used in conjunction with NMR to give more accurate structure determination of large proteins. While it is difficult to prove a model is correct with small angle scattering, it is possible to show that a proposed model is incorrect or incomplete.
• Characterisation of multi-domain proteins. Ab initio shape restoration is now possible using modern algorithms and large scale computing resources to provide information about the overall space envelope. When additional information is available, for example a crystallographic structure, for a subcomponent(s) of a modular protein or complex, it is possible to use a combination of ab initio shape restoration and rigid body refinement to model the structure of the protein.
• Modelling multi-subunit complexes. When significant conformation changes are associated with ligand binding, it is possible to monitor these changes in overall shape of the protein in the presence and absence of a ligand using SAXS.
• Flexible proteins. Protein SAXS data represent an average of all structures present in the solution throughout the measurement time. As such, it is possible to investigate the average solution structure of flexible proteins.
B21 is a dedicated solution state Small Angle X-ray Scattering (SAXS) beam line that can accommodate a wide variety of aqueous-based sample. B21 utilises a bending magnet source generating 1011 photons/sec directly to the sample.
The sensitivity and ability to perform very accurate data correction allow measurements over a range of concentration measurements, with high quality data achieved from very dilute samples (see plot below showing bioSAXS measurements of a dilution series of BSA).
Two data collection modes are currently available; bioSAXS robot and size exclusion chromatography SAXS (SEC-SAXS).
bioSAXS robot model
The current main sample environment on B21 is the EMBL-designed Arinax BioSAXS robot. This allows scientists to access high-throughput solution scattering, a technique of increasing importance to structural biologists. It may be possible to utilise the BioSAXS robot for low viscosity non-biological solutions (by prior arrangement only). The robot uses 96 well plates to hold samples - we can supply these and you may also use PCR strips if required. These samples are then delivered by the robot to a fixed glass capilliary inside the exposure unit which is held under vacuum to reduce background. The samples can be refrigerated while awaiting data collection if necessary.
SEC-SAXS mode
Inline SEC-SAXS is provided by an Agilent 1200 HPLC system. We offer several columns that include the 2.4 mL Superdex 200 (GE Healthcare) and the 4.6 mL KW-402, -403 and -404 (Shodex) columns. Superose columns operate at comparatively lower pressures, largely composed of conjugated sugars whereas the Shodex columns are higher pressure columns composed of silica. Samples are measured in a specialized cell with ~500x lower background than the bioSAXS robot cell. Please contact us for further details regarding column performance and expected dilution.
The integration of SEC-SAXS
One of the major problems faced during bioSAXS experiments is the monodispersity of the target protein. Even tiny amounts of impurities or protein aggregation caused from transport of sample can dramatically affect the quality of the scattering curves. The development of a high-performance liquid chromatography (HPLC) sample environment for solution scattering (SEC-SAXS) that couples the use of established biological separation techniques to the scattering experiment itself have helped resolve this issue. Work is ongoing at Diamond to establish a standardised system to enable high throughput of samples both in terms of sampling and software for SEC-SAXS.
BioSAXS for Flexible Proteins
BioSAXS for Macromolecular Complexes
BioSAXS for Ligand Binding
BioSAXS for Particulate Suspensions
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