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Brownian dynamics simulation of protofilament relaxation during rapid freezing

Electron cryo-microscopy (Cryo-EM) is a powerful method for visualizing biological objects with up to near-angstrom resolution. Instead of chemical fixation, the method relies on very rapid freezing to immobilize the sample. Under these conditions, crystalline ice does not have time to form and dist...

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Autores principales: Ulyanov, Evgeniy V., Vinogradov, Dmitrii S., McIntosh, J. Richard, Gudimchuk, Nikita B.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880439/
https://www.ncbi.nlm.nih.gov/pubmed/33577570
http://dx.doi.org/10.1371/journal.pone.0247022
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author Ulyanov, Evgeniy V.
Vinogradov, Dmitrii S.
McIntosh, J. Richard
Gudimchuk, Nikita B.
author_facet Ulyanov, Evgeniy V.
Vinogradov, Dmitrii S.
McIntosh, J. Richard
Gudimchuk, Nikita B.
author_sort Ulyanov, Evgeniy V.
collection PubMed
description Electron cryo-microscopy (Cryo-EM) is a powerful method for visualizing biological objects with up to near-angstrom resolution. Instead of chemical fixation, the method relies on very rapid freezing to immobilize the sample. Under these conditions, crystalline ice does not have time to form and distort structure. For many practical applications, the rate of cooling is fast enough to consider sample immobilization instantaneous, but in some cases, a more rigorous analysis of structure relaxation during freezing could be essential. This difficult yet important problem has been significantly under-reported in the literature, despite spectacular recent developments in Cryo-EM. Here we use Brownian dynamics modeling to examine theoretically the possible effects of cryo-immobilization on the apparent shapes of biological polymers. The main focus of our study is on tubulin protofilaments. These structures are integral parts of microtubules, which in turn are key elements of the cellular skeleton, essential for intracellular transport, maintenance of cell shape, cell division and migration. We theoretically examine the extent of protofilament relaxation within the freezing time as a function of the cooling rate, the filament’s flexural rigidity, and the effect of cooling on water’s viscosity. Our modeling suggests that practically achievable cooling rates are not rapid enough to capture tubulin protofilaments in conformations that are incompletely relaxed, suggesting that structures seen by cryo-EM are good approximations to physiological shapes. This prediction is confirmed by our analysis of curvatures of tubulin protofilaments, using samples, prepared and visualized with a variety of methods. We find, however, that cryofixation may capture incompletely relaxed shapes of more flexible polymers, and it may affect Cryo-EM-based measurements of their persistence lengths. This analysis will be valuable for understanding of structures of different types of biopolymers, observed with Cryo-EM.
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spelling pubmed-78804392021-02-19 Brownian dynamics simulation of protofilament relaxation during rapid freezing Ulyanov, Evgeniy V. Vinogradov, Dmitrii S. McIntosh, J. Richard Gudimchuk, Nikita B. PLoS One Research Article Electron cryo-microscopy (Cryo-EM) is a powerful method for visualizing biological objects with up to near-angstrom resolution. Instead of chemical fixation, the method relies on very rapid freezing to immobilize the sample. Under these conditions, crystalline ice does not have time to form and distort structure. For many practical applications, the rate of cooling is fast enough to consider sample immobilization instantaneous, but in some cases, a more rigorous analysis of structure relaxation during freezing could be essential. This difficult yet important problem has been significantly under-reported in the literature, despite spectacular recent developments in Cryo-EM. Here we use Brownian dynamics modeling to examine theoretically the possible effects of cryo-immobilization on the apparent shapes of biological polymers. The main focus of our study is on tubulin protofilaments. These structures are integral parts of microtubules, which in turn are key elements of the cellular skeleton, essential for intracellular transport, maintenance of cell shape, cell division and migration. We theoretically examine the extent of protofilament relaxation within the freezing time as a function of the cooling rate, the filament’s flexural rigidity, and the effect of cooling on water’s viscosity. Our modeling suggests that practically achievable cooling rates are not rapid enough to capture tubulin protofilaments in conformations that are incompletely relaxed, suggesting that structures seen by cryo-EM are good approximations to physiological shapes. This prediction is confirmed by our analysis of curvatures of tubulin protofilaments, using samples, prepared and visualized with a variety of methods. We find, however, that cryofixation may capture incompletely relaxed shapes of more flexible polymers, and it may affect Cryo-EM-based measurements of their persistence lengths. This analysis will be valuable for understanding of structures of different types of biopolymers, observed with Cryo-EM. Public Library of Science 2021-02-12 /pmc/articles/PMC7880439/ /pubmed/33577570 http://dx.doi.org/10.1371/journal.pone.0247022 Text en © 2021 Ulyanov et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Ulyanov, Evgeniy V.
Vinogradov, Dmitrii S.
McIntosh, J. Richard
Gudimchuk, Nikita B.
Brownian dynamics simulation of protofilament relaxation during rapid freezing
title Brownian dynamics simulation of protofilament relaxation during rapid freezing
title_full Brownian dynamics simulation of protofilament relaxation during rapid freezing
title_fullStr Brownian dynamics simulation of protofilament relaxation during rapid freezing
title_full_unstemmed Brownian dynamics simulation of protofilament relaxation during rapid freezing
title_short Brownian dynamics simulation of protofilament relaxation during rapid freezing
title_sort brownian dynamics simulation of protofilament relaxation during rapid freezing
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880439/
https://www.ncbi.nlm.nih.gov/pubmed/33577570
http://dx.doi.org/10.1371/journal.pone.0247022
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