A generalizable scaffold-based approach for structure determination of RNAs by cryo-EM
© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research..
Single-particle cryo-electron microscopy (cryo-EM) can reveal the structures of large and often dynamic molecules, but smaller biomolecules (≤50 kDa) remain challenging targets due to their intrinsic low signal to noise ratio. Methods to help resolve small proteins have been applied but development of similar approaches to aid in structural determination of small, structured RNA elements have lagged. Here, we present a scaffold-based approach that we used to recover maps of sub-25 kDa RNA domains to 4.5-5.0 Å. While lacking the detail of true high-resolution maps, these maps are suitable for model building and preliminary structure determination. We demonstrate this method helped faithfully recover the structure of several RNA elements of known structure, and that it promises to be generalized to other RNAs without disturbing their native fold. This approach may streamline the sample preparation process and reduce the optimization required for data collection. This first-generation scaffold approach provides a robust system to aid in RNA structure determination by cryo-EM and lays the groundwork for further scaffold optimization to achieve higher resolution.
| Errataetall: | |
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| Medienart: |
E-Artikel |
| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:51 |
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| Enthalten in: |
Nucleic acids research - 51(2023), 20 vom: 10. Nov., Seite e100 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Langeberg, Conner J [VerfasserIn] |
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| Links: |
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| Themen: |
63231-63-0 |
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| Anmerkungen: |
Date Completed 15.11.2023 Date Revised 11.04.2024 published: Print UpdateOf: bioRxiv. 2023 Jul 06;:. - PMID 37461535 Citation Status MEDLINE |
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| doi: |
10.1093/nar/gkad784 |
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| funding: |
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| 500 | |a UpdateOf: bioRxiv. 2023 Jul 06;:. - PMID 37461535 | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a © The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research. | ||
| 520 | |a Single-particle cryo-electron microscopy (cryo-EM) can reveal the structures of large and often dynamic molecules, but smaller biomolecules (≤50 kDa) remain challenging targets due to their intrinsic low signal to noise ratio. Methods to help resolve small proteins have been applied but development of similar approaches to aid in structural determination of small, structured RNA elements have lagged. Here, we present a scaffold-based approach that we used to recover maps of sub-25 kDa RNA domains to 4.5-5.0 Å. While lacking the detail of true high-resolution maps, these maps are suitable for model building and preliminary structure determination. We demonstrate this method helped faithfully recover the structure of several RNA elements of known structure, and that it promises to be generalized to other RNAs without disturbing their native fold. This approach may streamline the sample preparation process and reduce the optimization required for data collection. This first-generation scaffold approach provides a robust system to aid in RNA structure determination by cryo-EM and lays the groundwork for further scaffold optimization to achieve higher resolution | ||
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