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European Synchrotron Radiation Facility

6 ARTICLES PUBLISHED IN JoVE

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Biology

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography
Michael Heethoff 1, Lukas Helfen 2, Peter Cloetens 2
1Department of Evolutionary Biology of Invertebrates, University of Tubingen, 2European Synchrotron Radiation Facility

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7µm. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

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Biochemistry

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline
Martha E. Brennich 1, Adam R. Round 2,3, Stephanie Hutin 4
1Structural Biology Group, European Synchrotron Radiation Facility, 2European Molecular Biology Laboratory, 3School of Chemical and Physical Sciences, Keele University, 4Groupe de Microscopie Electronique et Méthodes, Institut de Biologie Structurale

The determination of the solution structure of a protein by small angle X-ray scattering (SAXS) requires monodisperse samples. Here, we present two possibilities to ensure minimal delays between sample preparation and data acquisition: online size-exclusion chromatography (SEC) and online ion-exchange chromatography (IEC).

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Biochemistry

Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect
Stephanie Hutin *1, Gianluca Santoni *1, Ulrich Zander 2, Nicolas Foos 1, Sylvain Aumonier 1, Guillaume Gotthard 1, Antoine Royant 1,3, Christoph Mueller-Dieckmann 1, Gordon Leonard 1
1European Synchrotron Radiation Facility, Structural Biology Group, 2European Molecular Biology Laboratory, 3Univ. Grenoble Alpes, CNRS, CEA, IBS (Institut de Biologie Structurale)

We present the use of the MeshAndCollect protocol to obtain a complete diffraction data set, for use in subsequent structure determination, composed of partial diffraction data sets collected from many small crystals of the fluorescent protein Cerulean.

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Biochemistry

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules
Stephanie Hutin 1, Bart Van Laer 1, Christoph Mueller-Dieckmann 1, Gordon Leonard 1, Didier Nurizzo 1, Matthew W. Bowler 2
1Structural Biology Group, European Synchrotron Radiation Facility, 2Grenoble Outstation, European Molecular Biology Laboratory

Here, we describe how to use the automated screening and data collection options available at some synchrotron beamlines. Scientists send cryocooled samples to the synchrotron, and the diffraction properties are screened, the data sets are collected and processed and, where possible, a structure solution is carried out—all without human intervention.

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Biochemistry

Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis
Caroline Bissardon 1, Solveig Reymond 1, Murielle Salomé 2, Lionel André 2, Sam Bayat 1, Peter Cloetens 2, Sylvain Bohic 1,2
1Inserm, UA7, Synchrotron Radiation for Biomedicine (STROBE), Université Grenoble Alpes, 2ID16A Beamline, ESRF, The European Synchrotron

Presented here is a protocol for cell culture on silicon nitride membranes and plunge-freezing prior to X-ray fluorescence imaging with a synchrotron cryogenic X-ray nanoprobe. When only room temperature nano-analysis is provided, the frozen samples can be further freeze-dried. These are critical steps to obtain information on the intracellular elemental composition.

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Biochemistry

Analysis of SEC-SAXS data via EFA deconvolution and Scatter
Mark D Tully *1, Nicolas Tarbouriech 2, Robert P Rambo 3, Stephanie Hutin *4
1European Synchrotron Radiation Facility Structural Biology Group, Structural Biology Group, 2Institut de Biologie Structurale, University Grenoble Alpes, CEA, CNRS, 3Diamond Light Source, 4Laboratoire de Physiologie Cellulaire and Végétale, Université Grenoble Alpes/CNRS/CEA/INRA/BIG

SEC-BioSAXS measurements of biological macromolecules are a standard approach for determining solution structure of macromolecules and their complexes. Here, we analyze SEC-BioSAXS data from two types of commonly encountered SEC traces—chromatograms with fully resolved and partially resolved peaks. We demonstrate the analysis and deconvolution using scatter and BioXTAS RAW.

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