Improvements in electron cryo-tomography towards understanding virus surfing
Electron cryo-tomography (cET), in combination with sub‑volume averaging (SVA), is a powerful technique for determining the structure of complex biological specimens that are not amenable to more traditional structure determination methods. In Chapter 1, SVA was used to achieve a sub-nanometre reso...
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| Format: | Thesis |
| Language: | English |
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2019
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| _version_ | 1797079208144404480 |
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| author | Prazak, V |
| author2 | Grünewald, K |
| author_facet | Grünewald, K Prazak, V |
| author_sort | Prazak, V |
| collection | OXFORD |
| description | Electron cryo-tomography (cET), in combination with sub‑volume averaging (SVA), is a powerful technique for determining the structure of complex biological specimens that are not amenable to more traditional structure determination methods. In Chapter 1, SVA was used to achieve a sub-nanometre resolution structure of Herpes simplex virus 1 glycoprotein B in its pre-fusion conformation. Nevertheless, several challenges still undermine the routine use of cET for structural cell biology. Imaging with an electron beam damages specimens, leading to localised warping of tomograms and reduced resolution of SVA maps if not compensated for. Using densities of biological structures within tomograms as fiducial markers is one solution to this problem. A software implementation of this concept, termed ‘Flexo’ and its application is demonstrated in Chapter 2. While this improves tomogram quality, other issues persist. The lack of a specific labelling technique for cET often limits the use of SVA to large and recognisable structures; correlation with super‑resolution cryo-fluorescence microscopy (cryo‑FM) is a promising solution. The primary drawback of this technique has been devitrification induced by the high laser intensities typically required. Chapter 3 describes Cryo-SOFI as a general solution to this problem, enabling substantial resolution improvement in cryo‑FM, and allowing cET to be performed in a correlative fashion. Chapter 4, explores the avenues of combining live-cell FM imaging with cET to study the transport of virus particles along cellular protrusions, called virus surfing. Events of surfing and static viruses can look alike in cET snapshots, making it necessary to link these high-resolution observations with their dynamic history. This led to the development of techniques towards live-CLEM: correlative live‑cell light and electron microscopy, which also revealed that cells undergo major structural changes during the preparation for cryogenic preservation. Overall, the work presented in this thesis presents a number of promising solutions to overcome some major obstacles for cET to fulfil its full potential for in situ structural cell biology.
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| first_indexed | 2024-03-07T00:42:29Z |
| format | Thesis |
| id | oxford-uuid:8389954f-99ab-40c8-a873-6bb0a175d65e |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T00:42:29Z |
| publishDate | 2019 |
| record_format | dspace |
| spelling | oxford-uuid:8389954f-99ab-40c8-a873-6bb0a175d65e2022-03-26T21:44:46ZImprovements in electron cryo-tomography towards understanding virus surfingThesishttp://purl.org/coar/resource_type/c_db06uuid:8389954f-99ab-40c8-a873-6bb0a175d65eMembrane fusionElectron Cryo-TomographyCryo-Fluorescence microscopyHerpes simplex virusStructural biologyVirologyMouse leukemia virusesEnglishHyrax Deposit2019Prazak, VGrünewald, KHuiskonen, JHeumann, JElectron cryo-tomography (cET), in combination with sub‑volume averaging (SVA), is a powerful technique for determining the structure of complex biological specimens that are not amenable to more traditional structure determination methods. In Chapter 1, SVA was used to achieve a sub-nanometre resolution structure of Herpes simplex virus 1 glycoprotein B in its pre-fusion conformation. Nevertheless, several challenges still undermine the routine use of cET for structural cell biology. Imaging with an electron beam damages specimens, leading to localised warping of tomograms and reduced resolution of SVA maps if not compensated for. Using densities of biological structures within tomograms as fiducial markers is one solution to this problem. A software implementation of this concept, termed ‘Flexo’ and its application is demonstrated in Chapter 2. While this improves tomogram quality, other issues persist. The lack of a specific labelling technique for cET often limits the use of SVA to large and recognisable structures; correlation with super‑resolution cryo-fluorescence microscopy (cryo‑FM) is a promising solution. The primary drawback of this technique has been devitrification induced by the high laser intensities typically required. Chapter 3 describes Cryo-SOFI as a general solution to this problem, enabling substantial resolution improvement in cryo‑FM, and allowing cET to be performed in a correlative fashion. Chapter 4, explores the avenues of combining live-cell FM imaging with cET to study the transport of virus particles along cellular protrusions, called virus surfing. Events of surfing and static viruses can look alike in cET snapshots, making it necessary to link these high-resolution observations with their dynamic history. This led to the development of techniques towards live-CLEM: correlative live‑cell light and electron microscopy, which also revealed that cells undergo major structural changes during the preparation for cryogenic preservation. Overall, the work presented in this thesis presents a number of promising solutions to overcome some major obstacles for cET to fulfil its full potential for in situ structural cell biology. |
| spellingShingle | Membrane fusion Electron Cryo-Tomography Cryo-Fluorescence microscopy Herpes simplex virus Structural biology Virology Mouse leukemia viruses Prazak, V Improvements in electron cryo-tomography towards understanding virus surfing |
| title | Improvements in electron cryo-tomography towards understanding virus surfing |
| title_full | Improvements in electron cryo-tomography towards understanding virus surfing |
| title_fullStr | Improvements in electron cryo-tomography towards understanding virus surfing |
| title_full_unstemmed | Improvements in electron cryo-tomography towards understanding virus surfing |
| title_short | Improvements in electron cryo-tomography towards understanding virus surfing |
| title_sort | improvements in electron cryo tomography towards understanding virus surfing |
| topic | Membrane fusion Electron Cryo-Tomography Cryo-Fluorescence microscopy Herpes simplex virus Structural biology Virology Mouse leukemia viruses |
| work_keys_str_mv | AT prazakv improvementsinelectroncryotomographytowardsunderstandingvirussurfing |