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Cryo-electron microscopy of an extremely halophilic microbe: technical aspects
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-...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer Japan
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5329092/ https://www.ncbi.nlm.nih.gov/pubmed/28050645 http://dx.doi.org/10.1007/s00792-016-0912-0 |
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author | Bollschweiler, Daniel Schaffer, Miroslava Lawrence, C. Martin Engelhardt, Harald |
author_facet | Bollschweiler, Daniel Schaffer, Miroslava Lawrence, C. Martin Engelhardt, Harald |
author_sort | Bollschweiler, Daniel |
collection | PubMed |
description | 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00792-016-0912-0) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5329092 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Springer Japan |
record_format | MEDLINE/PubMed |
spelling | pubmed-53290922017-03-22 Cryo-electron microscopy of an extremely halophilic microbe: technical aspects Bollschweiler, Daniel Schaffer, Miroslava Lawrence, C. Martin Engelhardt, Harald Extremophiles Original Paper 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00792-016-0912-0) contains supplementary material, which is available to authorized users. Springer Japan 2017-01-03 2017 /pmc/articles/PMC5329092/ /pubmed/28050645 http://dx.doi.org/10.1007/s00792-016-0912-0 Text en © The Author(s) 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. |
spellingShingle | Original Paper Bollschweiler, Daniel Schaffer, Miroslava Lawrence, C. Martin Engelhardt, Harald Cryo-electron microscopy of an extremely halophilic microbe: technical aspects |
title | Cryo-electron microscopy of an extremely halophilic microbe: technical aspects |
title_full | Cryo-electron microscopy of an extremely halophilic microbe: technical aspects |
title_fullStr | Cryo-electron microscopy of an extremely halophilic microbe: technical aspects |
title_full_unstemmed | Cryo-electron microscopy of an extremely halophilic microbe: technical aspects |
title_short | Cryo-electron microscopy of an extremely halophilic microbe: technical aspects |
title_sort | cryo-electron microscopy of an extremely halophilic microbe: technical aspects |
topic | Original Paper |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5329092/ https://www.ncbi.nlm.nih.gov/pubmed/28050645 http://dx.doi.org/10.1007/s00792-016-0912-0 |
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