Paul Scherrer Institute 6 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. Biochemistry Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip Raphaël de Wijn1,4, Kévin Rollet1,2, Vincent Olieric3, Oliver Hennig2, Nicola Thome1, Camille Noûs1, Caroline Paulus1, Bernard Lorber1, Heike Betat2, Mario Mörl2, Claude Sauter1 1Université de Strasbourg, Architecture et Réactivité de l’ARN, UPR 9002, CNRS, Institut de Biologie Moléculaire et Cellulaire, 2Biochemistry and Molecular Biology, Institute for Biochemistry, Leipzig University, 3Paul Scherrer Institute, Swiss Light Source, 4European XFEL GmbH A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature. Biochemistry Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments John H. Beale1, May E. Marsh1 1Swiss Light Source, Paul Scherrer Institut The aim of this article is to give the viewer a solid understanding of how to transform their small-volume, vapor-diffusion protocol, for growing large, single protein crystals, into a large-volume batch micro-crystallization method for serial crystallography. Biochemistry Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization Filip Pamula1,2, Jonas Mühle1, Alain Blanc3, Rony Nehmé4, Patricia C. Edwards4, Christopher G. Tate4, Ching-Ju Tsai1 1Laboratory of Biomolecular Research, Paul Scherrer Institute, 2Department of Biology, ETH Zürich, 3Center for Radiopharmaceutical Sciences, Paul Scherrer Institute, 4Laboratory of Molecular Biology, Medical Research Council This report describes screening of different detergents for preparing the visual GPCR, rhodopsin, and its complex with mini-Go. Biochemical methods characterizing the quality of the complex at different stages during purification are demonstrated. This protocol can be generalized to other membrane protein complexes for their future structural studies. Chemistry Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers Daniel James1, Tobias Weinert1, Petr Skopintsev1, Antonia Furrer1, Dardan Gashi1,2, Tomoyuki Tanaka3,4, Eriko Nango3,4, Przemyslaw Nogly1,5, Joerg Standfuss1 1Division of Biology and Chemistry - Laboratory for Biomolecular Research, Paul Scherrer Institut, 2Photon Science Division - SwissFEL, Paul Scherrer Institut, 3RIKEN SPring-8 Center, 4Department of Cell Biology, Graduate School of Medicine, Kyoto University, 5Department of Biology, ETH Zürich The success of a time-resolved serial femtosecond crystallography experiment is dependent on efficient sample delivery. Here, we describe protocols to optimize the extrusion of bacteriorhodopsin microcrystals from a high viscosity micro-extrusion injector. The methodology relies on sample homogenization with a novel three-way coupler and visualization with a high-speed camera. Chemistry A Practical Guide on Coupling a Scanning Mobility Sizer and Inductively Coupled Plasma Mass Spectrometer (SMPS-ICPMS) Mohamed Tarik1, Debora Foppiano1,2, Adrian Hess1,3, Christian Ludwig1,2 1Bioenergy and Catalysis Laboratory (LBK), Energy and Environment Research Division (ENE), Paul Scherrer Institute, 2Environmental Engineering Institute (IIE), School of Architecture, Civil and Environmental Engineering (ENAC), École; Polytechnique Fédérale de Lausanne (EPFL), 3Institute for Atmospheric and Climate Science ETH Zurich In this work a practical guide is provided, describing the different steps to establish the coupling of SMPS and ICPMS systems, and how to use them. Three descriptive examples are presented.
