Measuring the effects of ice thickness on resolution in single particle cryo-EM
Ice thickness is a critical parameter in single particle cryo-EM – too thin ice can break during imaging or exclude the sample of interest, while ice that is too thick contributes to more inelastic scattering that precludes obtaining high resolution reconstructions. Here we present the practical eff...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2023-01-01
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| Series: | Journal of Structural Biology: X |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2590152423000016 |
| _version_ | 1797798650825408512 |
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| author | Kasahun Neselu Bing Wang William J. Rice Clinton S. Potter Bridget Carragher Eugene Y.D. Chua |
| author_facet | Kasahun Neselu Bing Wang William J. Rice Clinton S. Potter Bridget Carragher Eugene Y.D. Chua |
| author_sort | Kasahun Neselu |
| collection | DOAJ |
| description | Ice thickness is a critical parameter in single particle cryo-EM – too thin ice can break during imaging or exclude the sample of interest, while ice that is too thick contributes to more inelastic scattering that precludes obtaining high resolution reconstructions. Here we present the practical effects of ice thickness on resolution, and the influence of energy filters, accelerating voltage, or detector mode. We collected apoferritin data with a wide range of ice thicknesses on three microscopes with different instrumentation and settings. We show that on a 300 kV microscope, using a 20 eV energy filter slit has a greater effect on improving resolution in thicker ice; that operating at 300 kV instead of 200 kV accelerating voltage provides significant resolution improvements at an ice thickness above 150 nm; and that on a 200 kV microscope using a detector operating in super resolution mode enables good reconstructions for up to 200 nm ice thickness, while collecting in counting instead of linear mode leads to improvements in resolution for ice of 50–150 nm thickness. Our findings can serve as a guide for users seeking to optimize data collection or sample preparation routines for both single particle and in situ cryo-EM. We note that most in situ data collection is done on samples in a range of ice thickness above 150 nm so these results may be especially relevant to that community. |
| first_indexed | 2024-03-13T04:07:02Z |
| format | Article |
| id | doaj.art-216ec6425afd4e4499642356c3da4b60 |
| institution | Directory Open Access Journal |
| issn | 2590-1524 |
| language | English |
| last_indexed | 2024-03-13T04:07:02Z |
| publishDate | 2023-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Structural Biology: X |
| spelling | doaj.art-216ec6425afd4e4499642356c3da4b602023-06-21T06:59:06ZengElsevierJournal of Structural Biology: X2590-15242023-01-017100085Measuring the effects of ice thickness on resolution in single particle cryo-EMKasahun Neselu0Bing Wang1William J. Rice2Clinton S. Potter3Bridget Carragher4Eugene Y.D. Chua5Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USACryo-Electron Microscopy Core, New York University Grossman School of Medicine, New York, NY, USACryo-Electron Microscopy Core, New York University Grossman School of Medicine, New York, NY, USA; Department of Cell Biology, New York University Grossman School of Medicine, New York, NY, USASimons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USASimons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA; Corresponding authors.Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA; Corresponding authors.Ice thickness is a critical parameter in single particle cryo-EM – too thin ice can break during imaging or exclude the sample of interest, while ice that is too thick contributes to more inelastic scattering that precludes obtaining high resolution reconstructions. Here we present the practical effects of ice thickness on resolution, and the influence of energy filters, accelerating voltage, or detector mode. We collected apoferritin data with a wide range of ice thicknesses on three microscopes with different instrumentation and settings. We show that on a 300 kV microscope, using a 20 eV energy filter slit has a greater effect on improving resolution in thicker ice; that operating at 300 kV instead of 200 kV accelerating voltage provides significant resolution improvements at an ice thickness above 150 nm; and that on a 200 kV microscope using a detector operating in super resolution mode enables good reconstructions for up to 200 nm ice thickness, while collecting in counting instead of linear mode leads to improvements in resolution for ice of 50–150 nm thickness. Our findings can serve as a guide for users seeking to optimize data collection or sample preparation routines for both single particle and in situ cryo-EM. We note that most in situ data collection is done on samples in a range of ice thickness above 150 nm so these results may be especially relevant to that community.http://www.sciencedirect.com/science/article/pii/S2590152423000016Cryo-EMIce thicknessSingle particle analysisEnergy filterHigh tensionResolution |
| spellingShingle | Kasahun Neselu Bing Wang William J. Rice Clinton S. Potter Bridget Carragher Eugene Y.D. Chua Measuring the effects of ice thickness on resolution in single particle cryo-EM Journal of Structural Biology: X Cryo-EM Ice thickness Single particle analysis Energy filter High tension Resolution |
| title | Measuring the effects of ice thickness on resolution in single particle cryo-EM |
| title_full | Measuring the effects of ice thickness on resolution in single particle cryo-EM |
| title_fullStr | Measuring the effects of ice thickness on resolution in single particle cryo-EM |
| title_full_unstemmed | Measuring the effects of ice thickness on resolution in single particle cryo-EM |
| title_short | Measuring the effects of ice thickness on resolution in single particle cryo-EM |
| title_sort | measuring the effects of ice thickness on resolution in single particle cryo em |
| topic | Cryo-EM Ice thickness Single particle analysis Energy filter High tension Resolution |
| url | http://www.sciencedirect.com/science/article/pii/S2590152423000016 |
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