Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography
Abstract Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of macromolecules in their biological environment. Eliminating biochemical isolation steps required b...
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| Format: | Article |
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Nature Portfolio
2021-03-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-021-22251-8 |
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| _version_ | 1849226079099682816 |
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| author | Jonathan Bouvette Hsuan-Fu Liu Xiaochen Du Ye Zhou Andrew P. Sikkema Juliana da Fonseca Rezende e Mello Bradley P. Klemm Rick Huang Roel M. Schaaper Mario J. Borgnia Alberto Bartesaghi |
| author_facet | Jonathan Bouvette Hsuan-Fu Liu Xiaochen Du Ye Zhou Andrew P. Sikkema Juliana da Fonseca Rezende e Mello Bradley P. Klemm Rick Huang Roel M. Schaaper Mario J. Borgnia Alberto Bartesaghi |
| author_sort | Jonathan Bouvette |
| collection | DOAJ |
| description | Abstract Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of macromolecules in their biological environment. Eliminating biochemical isolation steps required by other techniques, this method opens up the cell to in-situ structural studies. However, the need to compensate for errors in targeting introduced during mechanical navigation of the specimen significantly slows down tomographic data collection thus limiting its practical value. Here, we introduce protocols for tilt-series acquisition and processing that accelerate data collection speed by up to an order of magnitude and improve map resolution compared to existing approaches. We achieve this by using beam-image shift to multiply the number of areas imaged at each stage position, by integrating geometrical constraints during imaging to achieve high precision targeting, and by performing per-tilt astigmatic CTF estimation and data-driven exposure weighting to improve final map resolution. We validated our beam image-shift electron cryo-tomography (BISECT) approach by determining the structure of a low molecular weight target (~300 kDa) at 3.6 Å resolution where density for individual side chains is clearly resolved. |
| format | Article |
| id | doaj-art-984e7bb3da884354b7ff3bc7cde40146 |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2021-03-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-984e7bb3da884354b7ff3bc7cde401462025-08-24T11:39:53ZengNature PortfolioNature Communications2041-17232021-03-0112111110.1038/s41467-021-22251-8Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomographyJonathan Bouvette0Hsuan-Fu Liu1Xiaochen Du2Ye Zhou3Andrew P. Sikkema4Juliana da Fonseca Rezende e Mello5Bradley P. Klemm6Rick Huang7Roel M. Schaaper8Mario J. Borgnia9Alberto Bartesaghi10Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human ServicesDepartment of Biochemistry, Duke University School of MedicineDepartment of Computer Science, Duke UniversityDepartment of Computer Science, Duke UniversityEpigenetics & Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human ServicesGenome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human ServicesGenome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human ServicesLaboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Department of Health and Human ServicesGenome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human ServicesGenome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human ServicesDepartment of Biochemistry, Duke University School of MedicineAbstract Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of macromolecules in their biological environment. Eliminating biochemical isolation steps required by other techniques, this method opens up the cell to in-situ structural studies. However, the need to compensate for errors in targeting introduced during mechanical navigation of the specimen significantly slows down tomographic data collection thus limiting its practical value. Here, we introduce protocols for tilt-series acquisition and processing that accelerate data collection speed by up to an order of magnitude and improve map resolution compared to existing approaches. We achieve this by using beam-image shift to multiply the number of areas imaged at each stage position, by integrating geometrical constraints during imaging to achieve high precision targeting, and by performing per-tilt astigmatic CTF estimation and data-driven exposure weighting to improve final map resolution. We validated our beam image-shift electron cryo-tomography (BISECT) approach by determining the structure of a low molecular weight target (~300 kDa) at 3.6 Å resolution where density for individual side chains is clearly resolved.https://doi.org/10.1038/s41467-021-22251-8 |
| spellingShingle | Jonathan Bouvette Hsuan-Fu Liu Xiaochen Du Ye Zhou Andrew P. Sikkema Juliana da Fonseca Rezende e Mello Bradley P. Klemm Rick Huang Roel M. Schaaper Mario J. Borgnia Alberto Bartesaghi Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography Nature Communications |
| title | Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography |
| title_full | Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography |
| title_fullStr | Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography |
| title_full_unstemmed | Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography |
| title_short | Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography |
| title_sort | beam image shift accelerated data acquisition for near atomic resolution single particle cryo electron tomography |
| url | https://doi.org/10.1038/s41467-021-22251-8 |
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