AgarFix: Simple and accessible stabilization of challenging single-particle cryo-EM specimens through crosslinking in a matrix of agar

Klaudia Adamus, Sarah N. Le, Hans Elmlund, Marion Boudes, Dominika Elmlund

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Cryogenic electron microscopy (cryo-EM) allows structure determination of macromolecular assemblies that have resisted other structural biology approaches because of their size and heterogeneity. These challenging multi-protein targets are typically susceptible to dissociation and/or denaturation upon cryo-EM grid preparation, and often require crosslinking prior to freezing. Several approaches for gentle on-column or in-tube crosslinking have been developed. On-column crosslinking is not widely applicable because of the poor separation properties of gel filtration techniques. In-tube crosslinking frequently causes sample aggregation and/or precipitation. Gradient-based crosslinking through the GraFix method is more robust, but very time-consuming and necessitates specialised expensive equipment. Furthermore, removal of the glycerol typically involves significant sample loss and may cause destabilization detrimental to the sample quality. Here, we introduce an alternative procedure: AgarFix (Agarose Fixation). The sample is embedded in an agarose matrix that keeps the molecules separated, thus preventing formation of aggregates upon cross-inking. Gentle crosslinking is accomplished by diffusion of the cross-linker into the agarose drop. The sample is recovered by diffusion or electroelution and can readily be used for cryo-EM specimen preparation. AgarFix requires minimal equipment and basic lab experience, making it widely accessible to the cryo-EM community.

Original languageEnglish
Pages (from-to)327-331
Number of pages5
JournalJournal of Structural Biology
Volume207
Issue number3
DOIs
Publication statusPublished - 1 Sep 2019

Keywords

  • Crosslinking
  • Cryogenic electron microscopy
  • Multi-protein assemblies

Cite this

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title = "AgarFix: Simple and accessible stabilization of challenging single-particle cryo-EM specimens through crosslinking in a matrix of agar",
abstract = "Cryogenic electron microscopy (cryo-EM) allows structure determination of macromolecular assemblies that have resisted other structural biology approaches because of their size and heterogeneity. These challenging multi-protein targets are typically susceptible to dissociation and/or denaturation upon cryo-EM grid preparation, and often require crosslinking prior to freezing. Several approaches for gentle on-column or in-tube crosslinking have been developed. On-column crosslinking is not widely applicable because of the poor separation properties of gel filtration techniques. In-tube crosslinking frequently causes sample aggregation and/or precipitation. Gradient-based crosslinking through the GraFix method is more robust, but very time-consuming and necessitates specialised expensive equipment. Furthermore, removal of the glycerol typically involves significant sample loss and may cause destabilization detrimental to the sample quality. Here, we introduce an alternative procedure: AgarFix (Agarose Fixation). The sample is embedded in an agarose matrix that keeps the molecules separated, thus preventing formation of aggregates upon cross-inking. Gentle crosslinking is accomplished by diffusion of the cross-linker into the agarose drop. The sample is recovered by diffusion or electroelution and can readily be used for cryo-EM specimen preparation. AgarFix requires minimal equipment and basic lab experience, making it widely accessible to the cryo-EM community.",
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AgarFix : Simple and accessible stabilization of challenging single-particle cryo-EM specimens through crosslinking in a matrix of agar. / Adamus, Klaudia; Le, Sarah N.; Elmlund, Hans; Boudes, Marion; Elmlund, Dominika.

In: Journal of Structural Biology, Vol. 207, No. 3, 01.09.2019, p. 327-331.

Research output: Contribution to journalArticleResearchpeer-review

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AU - Elmlund, Dominika

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