title: A Versatile System for High-Throughput In Situ X-ray Screening and Data Collection of Soluble and Membrane-Protein Crystals.
abstract: In recent years, in situ data collection has been a major focus of progress in protein crystallography. Here, we introduce the Mylar in situ method using Mylar-based sandwich plates that are inexpensive, easy to make and handle, and show significantly less background scattering than other setups. A variety of cognate holders for patches of Mylar in situ sandwich films corresponding to one or more wells makes the method robust and versatile, allows for storage and shipping of entire wells, and enables automated crystal imaging, screening, and goniometer-based X-ray diffraction data-collection at room temperature and under cryogenic conditions for soluble and membrane-protein crystals grown in or transferred to these plates. We validated the Mylar in situ method using crystals of the water-soluble proteins hen egg-white lysozyme and sperm whale myoglobin as well as the 7-transmembrane protein bacteriorhodopsin fromHaloquadratum walsbyi. In conjunction with current developments at synchrotrons, this approach promises high-resolution structural studies of membrane proteins to become faster and more routine.
authors.affiliation: Department of Biochemistry and Department of Molecular Genetics, University of Toronto , Toronto, Ontario M5S 1A8, Canada.
authors.forename: Jana
authors.initials: J
authors.lastname: Broecker
authors.affiliation: Department of Biochemistry and Department of Molecular Genetics, University of Toronto , Toronto, Ontario M5S 1A8, Canada.
authors.forename: Viviane
authors.initials: V
authors.lastname: Klingel
authors.affiliation: Department of Biochemistry and Department of Molecular Genetics, University of Toronto , Toronto, Ontario M5S 1A8, Canada.
authors.forename: Wei-Lin
authors.initials: WL
authors.lastname: Ou
authors.affiliation: Department of Biochemistry and Department of Molecular Genetics, University of Toronto , Toronto, Ontario M5S 1A8, Canada.
authors.forename: Aidin R
authors.initials: AR
authors.lastname: Balo
authors.affiliation: GM/CA at Advanced Photon Source, Argonne National Laboratory , Lemont, Illinois 60439, United States.
authors.forename: David J
authors.initials: DJ
authors.lastname: Kissick
authors.affiliation: GM/CA at Advanced Photon Source, Argonne National Laboratory , Lemont, Illinois 60439, United States.
authors.forename: Craig M
authors.initials: CM
authors.lastname: Ogata
authors.affiliation: Department of Biochemistry and Department of Molecular Genetics, University of Toronto , Toronto, Ontario M5S 1A8, Canada.
authors.forename: Anling
authors.initials: A
authors.lastname: Kuo
authors.affiliation: Department of Biochemistry and Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Department of Biochemistry and Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Oliver P
authors.initials: OP
authors.lastname: Ernst