title: Cryo-electron microscopy of an extremely halophilic microbe: technical aspects.
abstract: Most halophilic Archaea of the class Halobacteriaceae depend on the presence of several molar sodium chloride for growth and cell integrity. This poses problems for structural studies, particularly for electron microscopy, where the high salt concentration results in diminished contrast. Since cryo-electron microscopy of intact cells provides new insights into the cellular and molecular organization under close-to-live conditions, we evaluated strategies and conditions to make halophilic microbes available for investigations in situ. Halobacterium salinarum, the test organism for this study, usually grows at 4.3 M NaCl. Adaptation to lower concentrations and subsequent NaCl reduction via dialysis led to still vital cells at 3 M salt. A comprehensive evaluation of vitrification parameters, thinning of frozen cells by focused-ion-beam micromachining, and cryo-electron microscopy revealed that structural studies under high salt conditions are possible in situ.
authors.affiliation: Max-Planck-Institut für Biochemie, Am Klopferspitz 18, 82152, Martinsried, Germany.|Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: Daniel
authors.initials: D
authors.lastname: Bollschweiler
authors.affiliation: Max-Planck-Institut für Biochemie, Am Klopferspitz 18, 82152, Martinsried, Germany.
authors.forename: Miroslava
authors.initials: M
authors.lastname: Schaffer
authors.affiliation: Max-Planck-Institut für Biochemie, Am Klopferspitz 18, 82152, Martinsried, Germany.|Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, USA.
authors.forename: C Martin
authors.initials: CM
authors.lastname: Lawrence
authors.affiliation: Max-Planck-Institut für Biochemie, Am Klopferspitz 18, 82152, Martinsried, Germany. engelhar@biochem.mpg.de.
authors.forename: Harald
authors.initials: H
authors.lastname: Engelhardt
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods*; D006217:Halobacterium / Q000648:ultrastructure*; D012965:Sodium Chloride / Q000737:chemistry; D058989:Vitrification
chemical_list: D012965:Sodium Chloride
keywords: Cryo-electron tomography; Focused-ion-beam micromachining; Gas vesicles; Haloarchaea; Halobacterium salinarum; Vitrification