Diamond Light Source LTD View Institution's Website 9 articles published in JoVE Biochemistry A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline Adam D. Crawshaw1, Emma V. Beale1,2, Anna J. Warren1, Andrew Stallwood1,3, Graham Duller1, Jose Trincao1, Gwyndaf Evans1,4 1Diamond Light Source Ltd, Harwell Science and Innovation Campus, 2Paul Scherrer Institut, 3Central Laser Facility, Science and Technologies Facilities Council, Harwell Science and Innovation Campus, 4Rosalind Franklin Institute, Harwell Science and Innovation Campus The signal-to-noise ratio of data is one of the most important considerations in performing X-ray diffraction measurements from microcrystals. The VMXm beamline provides a low-noise environment and microbeam for such experiments. Here, we describe sample preparation methods for mounting and cooling microcrystals for VMXm and other microfocus macromolecular crystallography beamlines. Biology A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography Johannes Groen1, Andrea Sorrentino1, Lucía Aballe1, Robert Oliete1, Ricardo Valcárcel1, Chidinma Okolo2, Ilias Kounatidis2, Maria Harkiolaki2, Ana Joaquina Pérez-Berná1, Eva Pereiro1 1MISTRAL beamline, Alba Light Source, Cerdanyola del Valles, Spain, 2Beamline B24, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK Here, a protocol describing the sample preparation and data collection steps required in cryo soft X-ray tomography (SXT) to image the ultrastructure of whole cryo-preserved cells at a resolution of 25 nm half pitch, is presented. Biochemistry Fixed Target Serial Data Collection at Diamond Light Source Sam Horrell1, Danny Axford1, Nicholas E. Devenish1, Ali Ebrahim1, Michael A. Hough2, Darren A. Sherrell1,3, Selina L. S. Storm1,4, Ivo Tews5, Jonathan A. R. Worrall2, Robin L. Owen1 1Diamond Light Source, Harwell Science and Innovation Campus, 2School of Life Sciences, University of Essex, 3X-ray Science Division, Argonne National Laboratory, 4European Molecular Biology Laboratory, Hamburg Outstation c/o DESY, 5Biological Sciences, Institute for Life Sciences, University of Southampton We present a comprehensive guide to fixed target sample preparation, data collection, and data processing for serial synchrotron crystallography at Diamond beamline I24. Biochemistry Analysis of SEC-SAXS data via EFA deconvolution and Scatter Mark D Tully*1, Nicolas Tarbouriech2, Robert P Rambo3, 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. Biochemistry Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092 Simone Culurgioni1,2, Minzhe Tang1,2, David R. Hall1, Martin A. Walsh1,2 1Diamond Light Source, Harwell Science & Innovation Campus, 2Research Complex at Harwell, Harwell Science & Innovation Campus A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented. Biology Volume Segmentation and Analysis of Biological Materials Using SuRVoS (Super-region Volume Segmentation) Workbench Michele C. Darrow1, Imanol Luengo1,2, Mark Basham1, Matthew C. Spink1, Sarah Irvine1, Andrew P. French2, Alun W. Ashton1, Elizabeth M.H. Duke1 1Science Division, Harwell Science and Innovation Campus, Diamond Light Source, 2School of Computer Science, University of Nottingham Segmentation of three-dimensional data from many imaging techniques is a major bottleneck in analysis of complex biological systems. Here, we describe the use of SuRVoS Workbench to semi-automatically segment volumetric data at various length-scales using example datasets from cryo-electron tomography, cryo soft X-ray tomography, and phase contrast X-ray tomography techniques. Engineering Measurement of X-ray Beam Coherence along Multiple Directions Using 2-D Checkerboard Phase Grating Shashidhara Marathe1,2, Xianbo Shi1, Michael J. Wojcik1, Albert T. Macrander1, Lahsen Assoufid1 1X-ray Science Division, Advanced Photon Source, Argonne National Laboratory, 2Division of Science, Diamond Light Source Ltd. The measurement protocol and data analysis procedure are given for obtaining transverse coherence of a synchrotron radiation X-ray source along four directions simultaneously using a single 2-D checkerboard phase grating. This simple technique can be applied for complete transverse coherence characterization of X-ray sources and X-ray optics. Biology Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli Louise E. Bird1, Heather Rada1, Anil Verma1, Raphael Gasper2,3, James Birch4, Matthew Jennions4, Jan Lӧwe3, Isabel Moraes4, Raymond J. Owens1 1Oxford Protein Production Facility, Research Complex at Harwell, 2Protein Crystallography Group, Ruhr University, 3MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, 4Membrane Protein Laboratory, Diamond Light Source A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented. Engineering Angle-resolved Photoemission Spectroscopy At Ultra-low Temperatures Sergey V. Borisenko1, Volodymyr B. Zabolotnyy1, Alexander A. Kordyuk1,2, Danil V. Evtushinsky1, Timur K. Kim1,3, Emanuela Carleschi4, Bryan P. Doyle4, Rosalba Fittipaldi5, Mario Cuoco5, Antonio Vecchione5, Helmut Berger6 1Institute for Solid State Research, IFW-Dresden, 2Institute of Metal Physics of National Academy of Sciences of Ukraine, 3Diamond Light Source LTD, 4Department of Physics, University of Johannesburg, 5CNR-SPIN, and Dipartimento di Fisica "E. R. Caianiello", Università di Salerno, 6Institute of Physics of Complex Matter, École Polytechnique Fédérale de Lausanne The overall goal of this method is to determine the low-energy electronic structure of solids at ultra-low temperatures using Angle-Resolved Photoemission Spectroscopy with synchrotron radiation.