دورية أكاديمية
SPOT-RASTR-A cryo-EM specimen preparation technique that overcomes problems with preferred orientation and the air/water interface.
| العنوان: | SPOT-RASTR-A cryo-EM specimen preparation technique that overcomes problems with preferred orientation and the air/water interface. |
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| المؤلفون: | Esfahani BG; Institute of Molecular Biophysics, Florida State University, 91 Chieftan Way, Tallahassee, FL 32306, USA., Randolph PS; Institute of Molecular Biophysics, Florida State University, 91 Chieftan Way, Tallahassee, FL 32306, USA., Peng R; Institute of Molecular Biophysics, Florida State University, 91 Chieftan Way, Tallahassee, FL 32306, USA., Grant T; Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA.; Morgridge Institute for Research, 330 N Orchard Street, Madison, WI 53715, USA., Stroupe ME; Institute of Molecular Biophysics, Florida State University, 91 Chieftan Way, Tallahassee, FL 32306, USA.; Department of Biological Science, Florida State University, 319 Stadium Drive, Tallahassee, FL 32306, USA., Stagg SM; Institute of Molecular Biophysics, Florida State University, 91 Chieftan Way, Tallahassee, FL 32306, USA.; Department of Biological Science, Florida State University, 319 Stadium Drive, Tallahassee, FL 32306, USA. |
| المصدر: | PNAS nexus [PNAS Nexus] 2024 Aug 06; Vol. 3 (8), pp. pgae284. Date of Electronic Publication: 2024 Aug 06 (Print Publication: 2024). |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: Oxford University Press on behalf of the National Academy of Sciences Country of Publication: England NLM ID: 9918367777906676 Publication Model: eCollection Cited Medium: Internet ISSN: 2752-6542 (Electronic) Linking ISSN: 27526542 NLM ISO Abbreviation: PNAS Nexus Subsets: PubMed not MEDLINE |
| أسماء مطبوعة: | Original Publication: [Oxford] : Oxford University Press on behalf of the National Academy of Sciences, [2022]- |
| مستخلص: | In cryogenic electron microscopy (cryo-EM), specimen preparation remains a bottleneck despite recent advancements. Classical plunge freezing methods often result in issues like aggregation and preferred orientations at the air/water interface. Many alternative methods have been proposed, but there remains a lack a universal solution, and multiple techniques are often required for challenging samples. Here, we demonstrate the use of lipid nanotubes with nickel NTA headgroups as a platform for cryo-EM sample preparation. His-tagged specimens of interest are added to the tubules, and they can be frozen by conventional plunge freezing. We show that the nanotubes protect samples from the air/water interface and promote a wider range of orientations. The reconstruction of average subtracted tubular regions (RASTR) method allows for the removal of the nanotubule signal from the cryo-EM images resulting in isolated images of specimens of interest. Testing with β-galactosidase validates the method's ability to capture particles at lower concentrations, overcome preferred orientations, and achieve near-atomic resolution reconstructions. Since the nanotubules can be identified and targeted automatically at low magnification, the method enables fully automated data collection. Furthermore, the particles on the tubes can be automatically identified and centered using 2D classification enabling particle picking without requiring prior information. Altogether, our approach that we call specimen preparation on a tube RASTR holds promise for overcoming air-water interface and preferred orientation challenges and offers the potential for fully automated cryo-EM data collection and structure determination. (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.) |
| التعليقات: | Update of: bioRxiv. 2024 Jan 25:2024.01.24.577038. doi: 10.1101/2024.01.24.577038. (PMID: 38501120) |
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| معلومات مُعتمدة: | R35 GM139616 United States GM NIGMS NIH HHS |
| فهرسة مساهمة: | Keywords: automation; resolution anisotropy; vitreous ice |
| تواريخ الأحداث: | Date Created: 20240807 Latest Revision: 20240813 |
| رمز التحديث: | 20240813 |
| مُعرف محوري في PubMed: | PMC11303004 |
| DOI: | 10.1093/pnasnexus/pgae284 |
| PMID: | 39108302 |
| قاعدة البيانات: | MEDLINE |
Full Text Finder | تدمد: | 2752-6542 |
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| DOI: | 10.1093/pnasnexus/pgae284 |