title: Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline.
abstract: Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, particle size and shape, and surface-to-volume ratio of macromolecules. The technique is applicable to a very wide variety of solution conditions spanning a broad range of concentrations, pH values, ionic strengths, temperatures, additives, etc., but the sample is required to be monodisperse. This caveat led to the implementation of liquid chromatography systems on SAXS beamlines. Here, we describe the upstream integration of size-exclusion (SEC) and ion-exchange chromatography (IEC) on a beamline, different methods for optimal background subtraction, and data reduction. As an example, we describe how we use SEC- and IEC-SAXS on a fragment of the essential vaccinia virus protein D5, consisting of a D5N helicase domain. We determine its overall shape and molecular weight, showing the hexameric structure of the protein.
authors.affiliation: Structural Biology Group, European Synchrotron Radiation Facility.
authors.forename: Martha E
authors.initials: ME
authors.lastname: Brennich
authors.affiliation: European Molecular Biology Laboratory; School of Chemical and Physical Sciences, Keele University.
authors.forename: Adam R
authors.initials: AR
authors.lastname: Round
authors.affiliation: Groupe de Microscopie Electronique et Méthodes, Institut de Biologie Structurale; stephanie.hutin@ibs.fr.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Hutin
mesh_terms: D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D053838:Scattering, Small Angle; D014616:Vaccinia virus / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014961:X-Ray Diffraction
chemical_list: D011994:Recombinant Proteins; D014764:Viral Proteins; D004265:DNA Helicases