• Login
    View Item 
    •   SMARTech Home
    • Georgia Tech Theses and Dissertations
    • Georgia Tech Theses and Dissertations
    • View Item
    •   SMARTech Home
    • Georgia Tech Theses and Dissertations
    • Georgia Tech Theses and Dissertations
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Identifying key factors in two-dimensional crystal production and sample preparation for structure-function studies of membrane proteins by cryo-EM

    Thumbnail
    View/Open
    JOHNSON-DISSERTATION-2013.pdf (202.7Mb)
    Date
    2013-11-18
    Author
    Johnson, Matthew C.
    Metadata
    Show full item record
    Abstract
    Electron crystallography of two-dimensional crystals is a structure-determination method well suited to the study of membrane protein structure-function. Two-dimensional crystals consist of ordered arrays of protein within reconstituted lipid bilayers, an arrangement that mimics the natural membrane environment. In this work we describe our recent progress in the use of this method with three different proteins, each providing a window into a separate paradigm in the electron crystallographic pipeline. Specific crystallization conditions for human leukotriene C₄ synthase (LTC₄S) have previously been determined, but our continued refinement of purification and crystallization has identified a number of additional parameters that greatly affect crystal size and quality, and we have developed a protocol to rapidly and reproducibly grow large, non-mosaic crystals of LTC₄S. The human gamma-glutamyl carboxylase (GGCX) has also been crystallized, but is sensitive to cryo-EM sample preparation conditions and we present here the successful reproduction of crystallization and refinement of cryo-EM sample preparation conditions. Lastly, we describe our crystallization screens with the Vibrio cholerae sodium-pumping NADH:ubiquinone reductase complex (Na⁺-NQR), and identify the factors critical to membrane reconstitution of the complex, a necessary first step towards crystallization. We also describe a semi-quantitative crystal screening protocol we have developed that provides quick and accurate method to assess two- dimensional crystallization trials, and discuss some general observations in optimization of membrane protein purification and two-dimensional crystallization for electron crystallography.
    URI
    http://hdl.handle.net/1853/52974
    Collections
    • Georgia Tech Theses and Dissertations [23877]
    • School of Biology Theses and Dissertations [464]

    Browse

    All of SMARTechCommunities & CollectionsDatesAuthorsTitlesSubjectsTypesThis CollectionDatesAuthorsTitlesSubjectsTypes

    My SMARTech

    Login

    Statistics

    View Usage StatisticsView Google Analytics Statistics
    facebook instagram twitter youtube
    • My Account
    • Contact us
    • Directory
    • Campus Map
    • Support/Give
    • Library Accessibility
      • About SMARTech
      • SMARTech Terms of Use
    Georgia Tech Library266 4th Street NW, Atlanta, GA 30332
    404.894.4500
    • Emergency Information
    • Legal and Privacy Information
    • Human Trafficking Notice
    • Accessibility
    • Accountability
    • Accreditation
    • Employment
    © 2020 Georgia Institute of Technology