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Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

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 spanni...

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Autores principales: Brennich, Martha E., Round, Adam R., Hutin, Stephanie
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MyJove Corporation 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409194/
https://www.ncbi.nlm.nih.gov/pubmed/28117806
http://dx.doi.org/10.3791/54861
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author Brennich, Martha E.
Round, Adam R.
Hutin, Stephanie
author_facet Brennich, Martha E.
Round, Adam R.
Hutin, Stephanie
author_sort Brennich, Martha E.
collection PubMed
description 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.
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spelling pubmed-54091942017-05-12 Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline Brennich, Martha E. Round, Adam R. Hutin, Stephanie J Vis Exp Biochemistry 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. MyJove Corporation 2017-01-05 /pmc/articles/PMC5409194/ /pubmed/28117806 http://dx.doi.org/10.3791/54861 Text en Copyright © 2017, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/
spellingShingle Biochemistry
Brennich, Martha E.
Round, Adam R.
Hutin, Stephanie
Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline
title Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline
title_full Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline
title_fullStr Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline
title_full_unstemmed Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline
title_short Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline
title_sort online size-exclusion and ion-exchange chromatography on a saxs beamline
topic Biochemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409194/
https://www.ncbi.nlm.nih.gov/pubmed/28117806
http://dx.doi.org/10.3791/54861
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