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Thicker Ice Improves the Integrity and Angular Distribution of CDC48A Hexamers on Cryo-EM Grids

Many cryogenic electron microscopy (cryo-EM) single particle analyses are constrained by the sample preparation step upon which aggregation, dissociation, and/or preferential orientation of particles can be introduced. Here, we report how we solved these problems in the case of CDC48A, a hexameric A...

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Autores principales: Huntington, Brandon, Zhao, Lingyun, Bron, Patrick, Shahul Hameed, Umar F., Arold, Stefan T., Qureshi, Bilal M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9247313/
https://www.ncbi.nlm.nih.gov/pubmed/35782862
http://dx.doi.org/10.3389/fmolb.2022.890390
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author Huntington, Brandon
Zhao, Lingyun
Bron, Patrick
Shahul Hameed, Umar F.
Arold, Stefan T.
Qureshi, Bilal M.
author_facet Huntington, Brandon
Zhao, Lingyun
Bron, Patrick
Shahul Hameed, Umar F.
Arold, Stefan T.
Qureshi, Bilal M.
author_sort Huntington, Brandon
collection PubMed
description Many cryogenic electron microscopy (cryo-EM) single particle analyses are constrained by the sample preparation step upon which aggregation, dissociation, and/or preferential orientation of particles can be introduced. Here, we report how we solved these problems in the case of CDC48A, a hexameric AAA ATPase from Arabidopsis thaliana. CDC48A hexamers are well preserved under negative staining conditions but disassemble during grid freezing using the classical blotting method. Vitrification of grids using the blot-free Chameleon method preserved the integrity of particles but resulted in their strong preferential orientation. We then used a strategy where we improved in parallel the purification of CDC48A and the conditions for cryo-EM data acquisition. Indeed, we noted that images taken from thicker ice presented an even distribution of intact particles with random orientations, but resulted in a lower image resolution. Consequently, in our case, distribution, orientation, image resolution, and the integrity of particles were tightly correlated with ice thickness. By combining the more homogeneous and stable CDC48A hexamers resulting from our improved purification protocol with an iterative search across different ice thicknesses, we identified an intermediate thickness that retained sufficiently high-resolution structural information while maintaining a complete distribution of particle orientations. Our approach may provide a simple, fast, and generally applicable strategy to record data of sufficient quality under standard laboratory and microscope settings. This method may be of particular value when time and resources are limited.
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spelling pubmed-92473132022-07-02 Thicker Ice Improves the Integrity and Angular Distribution of CDC48A Hexamers on Cryo-EM Grids Huntington, Brandon Zhao, Lingyun Bron, Patrick Shahul Hameed, Umar F. Arold, Stefan T. Qureshi, Bilal M. Front Mol Biosci Molecular Biosciences Many cryogenic electron microscopy (cryo-EM) single particle analyses are constrained by the sample preparation step upon which aggregation, dissociation, and/or preferential orientation of particles can be introduced. Here, we report how we solved these problems in the case of CDC48A, a hexameric AAA ATPase from Arabidopsis thaliana. CDC48A hexamers are well preserved under negative staining conditions but disassemble during grid freezing using the classical blotting method. Vitrification of grids using the blot-free Chameleon method preserved the integrity of particles but resulted in their strong preferential orientation. We then used a strategy where we improved in parallel the purification of CDC48A and the conditions for cryo-EM data acquisition. Indeed, we noted that images taken from thicker ice presented an even distribution of intact particles with random orientations, but resulted in a lower image resolution. Consequently, in our case, distribution, orientation, image resolution, and the integrity of particles were tightly correlated with ice thickness. By combining the more homogeneous and stable CDC48A hexamers resulting from our improved purification protocol with an iterative search across different ice thicknesses, we identified an intermediate thickness that retained sufficiently high-resolution structural information while maintaining a complete distribution of particle orientations. Our approach may provide a simple, fast, and generally applicable strategy to record data of sufficient quality under standard laboratory and microscope settings. This method may be of particular value when time and resources are limited. Frontiers Media S.A. 2022-06-17 /pmc/articles/PMC9247313/ /pubmed/35782862 http://dx.doi.org/10.3389/fmolb.2022.890390 Text en Copyright © 2022 Huntington, Zhao, Bron, Shahul Hameed, Arold and Qureshi. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Molecular Biosciences
Huntington, Brandon
Zhao, Lingyun
Bron, Patrick
Shahul Hameed, Umar F.
Arold, Stefan T.
Qureshi, Bilal M.
Thicker Ice Improves the Integrity and Angular Distribution of CDC48A Hexamers on Cryo-EM Grids
title Thicker Ice Improves the Integrity and Angular Distribution of CDC48A Hexamers on Cryo-EM Grids
title_full Thicker Ice Improves the Integrity and Angular Distribution of CDC48A Hexamers on Cryo-EM Grids
title_fullStr Thicker Ice Improves the Integrity and Angular Distribution of CDC48A Hexamers on Cryo-EM Grids
title_full_unstemmed Thicker Ice Improves the Integrity and Angular Distribution of CDC48A Hexamers on Cryo-EM Grids
title_short Thicker Ice Improves the Integrity and Angular Distribution of CDC48A Hexamers on Cryo-EM Grids
title_sort thicker ice improves the integrity and angular distribution of cdc48a hexamers on cryo-em grids
topic Molecular Biosciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9247313/
https://www.ncbi.nlm.nih.gov/pubmed/35782862
http://dx.doi.org/10.3389/fmolb.2022.890390
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