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European Molecular Biology Laboratory

10 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy
Benjamin Dale 1, Gregory P. McNerney 2, Deanna L. Thompson 2, Wolfgang Hübner 3, Thomas Huser 2, Benjamin K. Chen 1
1Division of Infectious Diseases, Department of Medicine, Immunology Institute, Mount Sinai School of Medicine , 2NSF Center for Biophotonics, University of California, Davis, 3Structural and Computational Biology Unit, European Molecular Biology Laboratory

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

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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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Immunology and Infection

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion
James J. Faust 1,2, Wayne Christenson 3,4,5, Kyle Doudrick 6, John Heddleston 7, Teng-Leong Chew 7, Marko Lampe 8, Arnat Balabiyev 1,2, Robert Ros 3,4,5, Tatiana P. Ugarova 1,2
1Center for Metabolic and Vascular Biology, Mayo Clinic, 2Molecular and Cellular Biosciences, School of Life Sciences, Arizona State University, 3Department of Physics, Arizona State University, 4Center for Biological Physics, Arizona State University, 5Biodesign Institute, Arizona State University, 6Department of Civil and Environmental Engineering and Earth Sciences, University of Notre Dame, 7Advanced Imaging Center, HHMI Janelia Research Campus, 8Advanced Light Microscopy Facility, European Molecular Biology Laboratory

This protocol describes the fabrication of optical-quality glass surfaces adsorbed with compounds containing long-chain hydrocarbons that can be used to monitor macrophage fusion of living specimens and enables super-resolution microscopy of fixed specimens.

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Bioengineering

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells
Rachel Santarella-Mellwig 1, Uta Haselmann 2, Nicole L. Schieber 1, Paul Walther 3, Yannick Schwab 1, Claude Antony 1, Ralf Bartenschlager 2,4, Inés Romero-Brey 2
1European Molecular Biology Laboratory, 2Department of Infectious Diseases, Molecular Virology, Heidelberg University, 3Central Facility for Electron Microscopy, Ulm University, 4Heidelberg Partner Site, German Center for Infection Research

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

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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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Chemistry

Exfoliation and Analysis of Large-area, Air-Sensitive Two-Dimensional Materials
Josh P. Thompson 1, M. Hasan Doha 1, Peter Murphy 1,2, Jin Hu 1,3, Hugh O.H. Churchill 1,3
1Department of Physics, University of Arkansas, 2Department of Physics, SUNY Geneseo, 3Institute for Nanoscale Science and Engineering, University of Arkansas

A method for exfoliating large thin flakes of air sensitive two-dimensional materials and safely transporting them for analysis outside of a glovebox is presented.

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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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Biology

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition
Felix Weis 1, Wim J. H. Hagen 1, Martin Schorb 2, Simone Mattei 1,3
1Structural and Computational Biology Unit, European Molecular Biology Laboratory, 2Electron Microscopy Core Facility, European Molecular Biology Laboratory, 3Imaging Centre, European Molecular Biology Laboratory

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.

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Biochemistry

The Automated Crystallography Pipelines at the EMBL HTX Facility in Grenoble
Irina Cornaciu 1,2, Raphael Bourgeas 1, Guillaume Hoffmann 1, Florine Dupeux 1,3, Anne-Sophie Humm 1, Vincent Mariaule 1, Andrea Pica 1,2, Damien Clavel 1,2, Gael Seroul 1,2, Peter Murphy 1, José Antonio Márquez 1,2
1European Molecular Biology Laboratory, 2ALPX S.A.S., 3Institut de Biologie Structurale

Here, we describe how to use the automated macromolecular crystallography pipelines for protein-to-structure, rapid ligand-protein complex analysis and large-scale fragment screening based on the CrystalDirect technology at the HTX Laboratory in EMBL Grenoble.

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Biology

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline
Ivana Čavka 1,2, Rory M. Power 3, Dietrich Walsh 3, Timo Zimmermann 1,3, Simone Köhler 1
1Cell Biology and Biophysics, European Molecular Biology Laboratory, 2Collaboration for joint PhD degree between EMBL and Heidelberg University, Faculty of Biosciences, 3EMBL Imaging Centre, European Molecular Biology Laboratory

Super-resolution microscopy can provide a detailed insight into the organization of components within the synaptonemal complex in meiosis. Here, we demonstrate a protocol to resolve individual proteins of the Caenorhabditis elegans synaptonemal complex.

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