CTFFIND5 provides improved insight into quality, tilt, and thickness of TEM samples
Images taken by transmission electron microscopes are usually affected by lens aberrations and image defocus, among other factors. These distortions can be modeled in reciprocal space using the contrast transfer function (CTF). Accurate estimation and correction of the CTF is essential for restoring...
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2024-12-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/97227 |
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| author | Johannes Elferich Lingli Kong Ximena Zottig Nikolaus Grigorieff |
| author_facet | Johannes Elferich Lingli Kong Ximena Zottig Nikolaus Grigorieff |
| author_sort | Johannes Elferich |
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| description | Images taken by transmission electron microscopes are usually affected by lens aberrations and image defocus, among other factors. These distortions can be modeled in reciprocal space using the contrast transfer function (CTF). Accurate estimation and correction of the CTF is essential for restoring the high-resolution signal in cryogenic electron microscopy (cryoEM). Previously, we described the implementation of algorithms for this task in the cisTEM software package (Grant et al., 2018). Here we show that taking sample characteristics, such as thickness and tilt, into account can improve CTF estimation. This is particularly important when imaging cellular samples, where measurement of sample thickness and geometry derived from accurate modeling of the Thon ring pattern helps judging the quality of the sample. This improved CTF estimation has been implemented in CTFFIND5, a new version of the cisTEM program CTFFIND. We evaluated the accuracy of these estimates using images of tilted aquaporin crystals and eukaryotic cells thinned by focused ion beam milling. We estimate that with micrographs of sufficient quality CTFFIND5 can measure sample tilt with an accuracy of 3° and sample thickness with an accuracy of 5 nm. |
| format | Article |
| id | doaj-art-fb382d0f34894af2965b0fbe5dcd01bf |
| institution | Kabale University |
| issn | 2050-084X |
| language | English |
| publishDate | 2024-12-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj-art-fb382d0f34894af2965b0fbe5dcd01bf2024-12-20T14:11:12ZengeLife Sciences Publications LtdeLife2050-084X2024-12-011310.7554/eLife.97227CTFFIND5 provides improved insight into quality, tilt, and thickness of TEM samplesJohannes Elferich0https://orcid.org/0000-0002-9911-706XLingli Kong1https://orcid.org/0000-0002-5808-2649Ximena Zottig2https://orcid.org/0000-0002-2344-5163Nikolaus Grigorieff3https://orcid.org/0000-0002-1506-909XRNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, United States; Howard Hughes Medical Institute, University of Massachusetts Chan Medical School, Worcester, United StatesRNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, United StatesRNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, United States; Howard Hughes Medical Institute, University of Massachusetts Chan Medical School, Worcester, United StatesRNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, United States; Howard Hughes Medical Institute, University of Massachusetts Chan Medical School, Worcester, United StatesImages taken by transmission electron microscopes are usually affected by lens aberrations and image defocus, among other factors. These distortions can be modeled in reciprocal space using the contrast transfer function (CTF). Accurate estimation and correction of the CTF is essential for restoring the high-resolution signal in cryogenic electron microscopy (cryoEM). Previously, we described the implementation of algorithms for this task in the cisTEM software package (Grant et al., 2018). Here we show that taking sample characteristics, such as thickness and tilt, into account can improve CTF estimation. This is particularly important when imaging cellular samples, where measurement of sample thickness and geometry derived from accurate modeling of the Thon ring pattern helps judging the quality of the sample. This improved CTF estimation has been implemented in CTFFIND5, a new version of the cisTEM program CTFFIND. We evaluated the accuracy of these estimates using images of tilted aquaporin crystals and eukaryotic cells thinned by focused ion beam milling. We estimate that with micrographs of sufficient quality CTFFIND5 can measure sample tilt with an accuracy of 3° and sample thickness with an accuracy of 5 nm.https://elifesciences.org/articles/97227cryoEMelectron microscopycontrast transfer functionsample quality |
| spellingShingle | Johannes Elferich Lingli Kong Ximena Zottig Nikolaus Grigorieff CTFFIND5 provides improved insight into quality, tilt, and thickness of TEM samples eLife cryoEM electron microscopy contrast transfer function sample quality |
| title | CTFFIND5 provides improved insight into quality, tilt, and thickness of TEM samples |
| title_full | CTFFIND5 provides improved insight into quality, tilt, and thickness of TEM samples |
| title_fullStr | CTFFIND5 provides improved insight into quality, tilt, and thickness of TEM samples |
| title_full_unstemmed | CTFFIND5 provides improved insight into quality, tilt, and thickness of TEM samples |
| title_short | CTFFIND5 provides improved insight into quality, tilt, and thickness of TEM samples |
| title_sort | ctffind5 provides improved insight into quality tilt and thickness of tem samples |
| topic | cryoEM electron microscopy contrast transfer function sample quality |
| url | https://elifesciences.org/articles/97227 |
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