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Design and implementation of suspended drop crystallization

We have developed a novel crystal growth method known as suspended drop crystallization. Unlike traditional methods, this technique involves mixing protein and precipitant directly on an electron microscopy grid without any additional support layers. The grid is then suspended within a crystallizati...

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Detalles Bibliográficos
Autores principales: Gillman, Cody, Nicolas, William J., Martynowycz, Michael W., Gonen, Tamir
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10081258/
https://www.ncbi.nlm.nih.gov/pubmed/37034794
http://dx.doi.org/10.1101/2023.03.28.534639
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author Gillman, Cody
Nicolas, William J.
Martynowycz, Michael W.
Gonen, Tamir
author_facet Gillman, Cody
Nicolas, William J.
Martynowycz, Michael W.
Gonen, Tamir
author_sort Gillman, Cody
collection PubMed
description We have developed a novel crystal growth method known as suspended drop crystallization. Unlike traditional methods, this technique involves mixing protein and precipitant directly on an electron microscopy grid without any additional support layers. The grid is then suspended within a crystallization chamber which we designed, allowing for vapor diffusion to occur from both sides of the drop. A UV transparent window above and below the grid enables the monitoring of crystal growth via light, UV, or fluorescence microscopy. Once crystals have formed, the grid can be removed and utilized for x-ray crystallography or microcrystal electron diffraction (MicroED) directly without having to manipulate the crystals. To demonstrate the efficacy of this method, we grew crystals of the enzyme proteinase K and determined its structure by MicroED following FIB/SEM milling to render the sample thin enough for cryoEM. Suspended drop crystallization overcomes many of the challenges associated with sample preparation, providing an alternative workflow for crystals embedded in viscous media, sensitive to mechanical stress, and/or suffering from preferred orientation on EM grids.
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spelling pubmed-100812582023-04-08 Design and implementation of suspended drop crystallization Gillman, Cody Nicolas, William J. Martynowycz, Michael W. Gonen, Tamir bioRxiv Article We have developed a novel crystal growth method known as suspended drop crystallization. Unlike traditional methods, this technique involves mixing protein and precipitant directly on an electron microscopy grid without any additional support layers. The grid is then suspended within a crystallization chamber which we designed, allowing for vapor diffusion to occur from both sides of the drop. A UV transparent window above and below the grid enables the monitoring of crystal growth via light, UV, or fluorescence microscopy. Once crystals have formed, the grid can be removed and utilized for x-ray crystallography or microcrystal electron diffraction (MicroED) directly without having to manipulate the crystals. To demonstrate the efficacy of this method, we grew crystals of the enzyme proteinase K and determined its structure by MicroED following FIB/SEM milling to render the sample thin enough for cryoEM. Suspended drop crystallization overcomes many of the challenges associated with sample preparation, providing an alternative workflow for crystals embedded in viscous media, sensitive to mechanical stress, and/or suffering from preferred orientation on EM grids. Cold Spring Harbor Laboratory 2023-03-28 /pmc/articles/PMC10081258/ /pubmed/37034794 http://dx.doi.org/10.1101/2023.03.28.534639 Text en https://creativecommons.org/licenses/by/4.0/This work is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) , which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use.
spellingShingle Article
Gillman, Cody
Nicolas, William J.
Martynowycz, Michael W.
Gonen, Tamir
Design and implementation of suspended drop crystallization
title Design and implementation of suspended drop crystallization
title_full Design and implementation of suspended drop crystallization
title_fullStr Design and implementation of suspended drop crystallization
title_full_unstemmed Design and implementation of suspended drop crystallization
title_short Design and implementation of suspended drop crystallization
title_sort design and implementation of suspended drop crystallization
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10081258/
https://www.ncbi.nlm.nih.gov/pubmed/37034794
http://dx.doi.org/10.1101/2023.03.28.534639
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