EMBL Heidelberg (European Molecular Biology Laboratory) View Institution's Website 10 articles published in JoVE Biology Deciphering High-Resolution 3D Chromatin Organization via Capture Hi-C Antonia Hauth1, Rafael Galupa1, Nicolas Servant2, Laura Villacorta1, Kai Hauschulz3, Joke Gerarda van Bemmel4, Agnese Loda1, Edith Heard1,5 1EMBL: European Molecular Biology Laboratory, 2Institut Curie, 3Agilent Technologies, 4Genmab BV, 5Collège de France This protocol describes the Capture Hi-C method used to characterize the 3D organization of megabased-sized targeted genomic regions at high-resolution, including boundaries of topologically associating domains (TADs) and long-range chromatin interactions between regulatory and other DNA sequence elements. Biology Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline Ivana Čavka1,2, Rory M. Power3, Dietrich Walsh3, Timo Zimmermann1,3, Simone Köhler1 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. Biology Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition Felix Weis1, Wim J. H. Hagen1, Martin Schorb2, Simone Mattei1,3 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. Bioengineering Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells Rachel Santarella-Mellwig1, Uta Haselmann2, Nicole L. Schieber1, Paul Walther3, Yannick Schwab1, Claude Antony1, Ralf Bartenschlager2,4, Inés Romero-Brey2 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. Chemistry Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography Yannig Gicquel*1,2, Robin Schubert*3,4,5, Svetlana Kapis3, Gleb Bourenkov6, Thomas Schneider6, Markus Perbandt3,4, Christian Betzel3,4,5, Henry N. Chapman1,2,4, Michael Heymann1,7 1Center for Free Electron Laser Science, DESY, 2Department of Physics, University of Hamburg, 3Institute for Biochemistry and Molecular Biology, Laboratory for Structural Biology of Infection and Inflammation, University of Hamburg, 4The Hamburg Center for Ultrafast Imaging, University of Hamburg, 5Integrated Biology Infrastructure Life-Science Facility at the European XFEL (XBI), 6European Molecular Biology Laboratory, EMBL c/o DESY, 7Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data. Immunology and Infection Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion James J. Faust1,2, Wayne Christenson3,4,5, Kyle Doudrick6, John Heddleston7, Teng-Leong Chew7, Marko Lampe8, Arnat Balabiyev1,2, Robert Ros3,4,5, Tatiana P. Ugarova1,2 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. Biochemistry Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline Martha E. Brennich1, Adam R. Round2,3, Stephanie Hutin4 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). Biology iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution Julian Konig1, Kathi Zarnack2, Gregor Rot3, Tomaz Curk3, Melis Kayikci1, Blaz Zupan3, Daniel J. Turner4, Nicholas M. Luscombe2, Jernej Ule1 1Laboratory of Molecular Biology, Medical Research Council - MRC, 2European Bioinformatics Institute, EMBL Heidelberg, 3Computer and Information Science, University of Ljubljana, 4Wellcome Trust Genome Campus, Wellcome Trust Sanger Institute The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein. Immunology and Infection Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy Benjamin Dale1, Gregory P. McNerney2, Deanna L. Thompson2, Wolfgang Hübner3, Thomas Huser2, Benjamin K. Chen1 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. Biology Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid Ann-Marie Lawrence1, Hüseyin Besir1 1Protein Expression and Purification Core Facility, EMBL Heidelberg A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.
