EMBL Heidelberg (European Molecular Biology Laboratory)

9 articles published in JoVE

• Biology

  
  Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline Ivana Čavka^1,2, Rory M. Power³, Dietrich Walsh³, Timo Zimmermann^1,3, Simone Köhler^{1 1}Cell 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 Mattei^{1,3 1}Structural 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 Bartenschlager^2,4, Inés Romero-Brey^{2 1}European 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 Perbandt^3,4, Christian Betzel^3,4,5, Henry N. Chapman^1,2,4, Michael Heymann^{1,7 1}Center 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. Faust^1,2, Wayne Christenson^3,4,5, Kyle Doudrick⁶, John Heddleston⁷, Teng-Leong Chew⁷, Marko Lampe⁸, Arnat Balabiyev^1,2, Robert Ros^3,4,5, Tatiana P. Ugarova^{1,2 1}Center 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. Round^2,3, Stephanie Hutin^{4 1}Structural 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 Ule^{1 1}Laboratory 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. Chen^{1 1}Division 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 Besir^{1 1}Protein 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.