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Direct deposition on pre-cooled grids: a new approach to sub-millisecond cryoEM grid preparation.

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Τίτλος: Direct deposition on pre-cooled grids: a new approach to sub-millisecond cryoEM grid preparation.
Συγγραφείς: Klebl DP; School of Biomedical Sciences, Faculty of Biomedical Sciences and Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds, United Kingdom., Kay RW; School of Mechanical Engineering, University of Leeds, Leeds, United Kingdom., McMillan S; School of Biomedical Sciences, Faculty of Biomedical Sciences and Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds, United Kingdom., Sobott F; School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds, United Kingdom., Kapur N; School of Mechanical Engineering, University of Leeds, Leeds, United Kingdom., Muench SP; School of Biomedical Sciences, Faculty of Biomedical Sciences and Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds, United Kingdom.
Πηγή: Acta crystallographica. Section D, Structural biology [Acta Crystallogr D Struct Biol] 2026 Sep 01; Vol. 82 (Pt 9), pp. 1056-1061. Date of Electronic Publication: 2026 Aug 11.
Τύπος έκδοσης: Journal Article
Γλώσσα: English
Στοιχεία περιοδικού: Publisher: John Wiley & Sons Country of Publication: United States NLM ID: 101676043 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 2059-7983 (Electronic) Linking ISSN: 20597983 NLM ISO Abbreviation: Acta Crystallogr D Struct Biol Subsets: MEDLINE
Imprint Name(s): Publication: <2018-> : Medford, MA : John Wiley & Sons
Original Publication: [Malden, MA] : John Wiley & Sons, [2016]-
Ιατρικοί όροι (MeSH): Cryoelectron Microscopy*/methods , Cryoelectron Microscopy*/instrumentation , Specimen Handling*/methods
Περίληψη: There has been a rapid expansion in new approaches to sample preparation for single-particle cryoEM. One drive has been around speeding up the process both for the application of time-resolved studies and in outrunning some of the negative effects which may come from the sample interacting with the air-water interface, such as degradation and preferred orientation. Here, we set out to significantly improve the speed of grid preparation by removing the plunging stage and directly spraying onto pre-cooled grids which are situated in the cryogen, allowing us to produce grids where two samples are mixed and frozen on the grid in less than 1 ms. We have previously shown a preliminary proof of principle with this new approach and here we provide a detailed description of the approach taken and the methodology behind it. This approach allows us to move beyond the millisecond timescale to open up more systems to time-resolved EM and potentially improve samples.
(open access.)
References: IUCrJ. 2019 Sep 05;6(Pt 6):1024-1031. (PMID: 31709058)
Faraday Discuss. 2022 Nov 8;240(0):55-66. (PMID: 35924676)
Biochem Soc Trans. 2024 Jun 26;52(3):1529-1537. (PMID: 38864435)
Nat Methods. 2025 Sep;22(9):1880-1886. (PMID: 40877464)
Elife. 2019 Apr 01;8:. (PMID: 30932812)
Front Mol Biosci. 2022 Jun 23;9:903148. (PMID: 35813832)
Acta Crystallogr D Struct Biol. 2020 Apr 1;76(Pt 4):340-349. (PMID: 32254058)
Structure. 2020 Nov 3;28(11):1238-1248.e4. (PMID: 32814033)
Acta Crystallogr D Struct Biol. 2019 Dec 1;75(Pt 12):1063-1070. (PMID: 31793900)
Nat Commun. 2024 Sep 4;15(1):7705. (PMID: 39231985)
J Vis Exp. 2021 Nov 6;(177):. (PMID: 34806700)
Nat Methods. 2021 May;18(5):463-471. (PMID: 33963356)
Proc Natl Acad Sci U S A. 2025 Oct 28;122(43):e2516900122. (PMID: 41123998)
Nat Commun. 2020 May 22;11(1):2563. (PMID: 32444637)
Faraday Discuss. 2022 Nov 8;240(0):33-43. (PMID: 35920384)
Nat Methods. 2014 Jun;11(6):649-52. (PMID: 24747813)
J Struct Biol X. 2019 Jan-Mar;1:. (PMID: 32285040)
Grant Information: United Kingdom WT_ Wellcome Trust; 198466/Z/15/Z United Kingdom WT_ Wellcome Trust; UKR11958 United Kingdom BB_ Biotechnology and Biological Sciences Research Council
Contributed Indexing: Keywords: cryoEM; structural biology; time-resolved cryoEM
Entry Date(s): Date Created: 20260811 Date Completed: 20260901 Latest Revision: 20260903
Update Code: 20260903
PubMed Central ID: PMC13532694
DOI: 10.1107/S2059798326007126
PMID: 42579337
Βάση Δεδομένων: MEDLINE
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  Data: Direct deposition on pre-cooled grids: a new approach to sub-millisecond cryoEM grid preparation.
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  Data: &lt;searchLink fieldCode=&quot;AU&quot; term=&quot;%22Klebl+DP%22&quot;&gt;Klebl DP&lt;/searchLink&gt;; School of Biomedical Sciences, Faculty of Biomedical Sciences and Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds, United Kingdom.&lt;br /&gt;&lt;searchLink fieldCode=&quot;AU&quot; term=&quot;%22Kay+RW%22&quot;&gt;Kay RW&lt;/searchLink&gt;; School of Mechanical Engineering, University of Leeds, Leeds, United Kingdom.&lt;br /&gt;&lt;searchLink fieldCode=&quot;AU&quot; term=&quot;%22McMillan+S%22&quot;&gt;McMillan S&lt;/searchLink&gt;; School of Biomedical Sciences, Faculty of Biomedical Sciences and Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds, United Kingdom.&lt;br /&gt;&lt;searchLink fieldCode=&quot;AU&quot; term=&quot;%22Sobott+F%22&quot;&gt;Sobott F&lt;/searchLink&gt;; School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds, United Kingdom.&lt;br /&gt;&lt;searchLink fieldCode=&quot;AU&quot; term=&quot;%22Kapur+N%22&quot;&gt;Kapur N&lt;/searchLink&gt;; School of Mechanical Engineering, University of Leeds, Leeds, United Kingdom.&lt;br /&gt;&lt;searchLink fieldCode=&quot;AU&quot; term=&quot;%22Muench+SP%22&quot;&gt;Muench SP&lt;/searchLink&gt;; School of Biomedical Sciences, Faculty of Biomedical Sciences and Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds, United Kingdom.
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  Data: There has been a rapid expansion in new approaches to sample preparation for single-particle cryoEM. One drive has been around speeding up the process both for the application of time-resolved studies and in outrunning some of the negative effects which may come from the sample interacting with the air-water interface, such as degradation and preferred orientation. Here, we set out to significantly improve the speed of grid preparation by removing the plunging stage and directly spraying onto pre-cooled grids which are situated in the cryogen, allowing us to produce grids where two samples are mixed and frozen on the grid in less than 1 ms. We have previously shown a preliminary proof of principle with this new approach and here we provide a detailed description of the approach taken and the methodology behind it. This approach allows us to move beyond the millisecond timescale to open up more systems to time-resolved EM and potentially improve samples.&lt;br /&gt; (open access.)
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  Data: IUCrJ. 2019 Sep 05;6(Pt 6):1024-1031. (PMID: &lt;searchLink fieldCode=&quot;PM&quot; term=&quot;%2231709058%22&quot;&gt;31709058)&lt;/searchLink&gt;&lt;br /&gt;Faraday Discuss. 2022 Nov 8;240(0):55-66. (PMID: &lt;searchLink fieldCode=&quot;PM&quot; term=&quot;%2235924676%22&quot;&gt;35924676)&lt;/searchLink&gt;&lt;br /&gt;Biochem Soc Trans. 2024 Jun 26;52(3):1529-1537. (PMID: &lt;searchLink fieldCode=&quot;PM&quot; term=&quot;%2238864435%22&quot;&gt;38864435)&lt;/searchLink&gt;&lt;br /&gt;Nat Methods. 2025 Sep;22(9):1880-1886. (PMID: &lt;searchLink fieldCode=&quot;PM&quot; term=&quot;%2240877464%22&quot;&gt;40877464)&lt;/searchLink&gt;&lt;br /&gt;Elife. 2019 Apr 01;8:. (PMID: &lt;searchLink fieldCode=&quot;PM&quot; term=&quot;%2230932812%22&quot;&gt;30932812)&lt;/searchLink&gt;&lt;br /&gt;Front Mol Biosci. 2022 Jun 23;9:903148. (PMID: &lt;searchLink fieldCode=&quot;PM&quot; term=&quot;%2235813832%22&quot;&gt;35813832)&lt;/searchLink&gt;&lt;br /&gt;Acta Crystallogr D Struct Biol. 2020 Apr 1;76(Pt 4):340-349. (PMID: &lt;searchLink fieldCode=&quot;PM&quot; term=&quot;%2232254058%22&quot;&gt;32254058)&lt;/searchLink&gt;&lt;br /&gt;Structure. 2020 Nov 3;28(11):1238-1248.e4. (PMID: &lt;searchLink fieldCode=&quot;PM&quot; term=&quot;%2232814033%22&quot;&gt;32814033)&lt;/searchLink&gt;&lt;br /&gt;Acta Crystallogr D Struct Biol. 2019 Dec 1;75(Pt 12):1063-1070. (PMID: &lt;searchLink fieldCode=&quot;PM&quot; term=&quot;%2231793900%22&quot;&gt;31793900)&lt;/searchLink&gt;&lt;br /&gt;Nat Commun. 2024 Sep 4;15(1):7705. (PMID: &lt;searchLink fieldCode=&quot;PM&quot; term=&quot;%2239231985%22&quot;&gt;39231985)&lt;/searchLink&gt;&lt;br /&gt;J Vis Exp. 2021 Nov 6;(177):. (PMID: &lt;searchLink fieldCode=&quot;PM&quot; term=&quot;%2234806700%22&quot;&gt;34806700)&lt;/searchLink&gt;&lt;br /&gt;Nat Methods. 2021 May;18(5):463-471. (PMID: &lt;searchLink fieldCode=&quot;PM&quot; term=&quot;%2233963356%22&quot;&gt;33963356)&lt;/searchLink&gt;&lt;br /&gt;Proc Natl Acad Sci U S A. 2025 Oct 28;122(43):e2516900122. (PMID: &lt;searchLink fieldCode=&quot;PM&quot; term=&quot;%2241123998%22&quot;&gt;41123998)&lt;/searchLink&gt;&lt;br /&gt;Nat Commun. 2020 May 22;11(1):2563. (PMID: &lt;searchLink fieldCode=&quot;PM&quot; term=&quot;%2232444637%22&quot;&gt;32444637)&lt;/searchLink&gt;&lt;br /&gt;Faraday Discuss. 2022 Nov 8;240(0):33-43. (PMID: &lt;searchLink fieldCode=&quot;PM&quot; term=&quot;%2235920384%22&quot;&gt;35920384)&lt;/searchLink&gt;&lt;br /&gt;Nat Methods. 2014 Jun;11(6):649-52. (PMID: &lt;searchLink fieldCode=&quot;PM&quot; term=&quot;%2224747813%22&quot;&gt;24747813)&lt;/searchLink&gt;&lt;br /&gt;J Struct Biol X. 2019 Jan-Mar;1:. (PMID: &lt;searchLink fieldCode=&quot;PM&quot; term=&quot;%2232285040%22&quot;&gt;32285040)&lt;/searchLink&gt;
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  Data: United Kingdom WT_ Wellcome Trust; 198466/Z/15/Z United Kingdom WT_ Wellcome Trust; UKR11958 United Kingdom BB_ Biotechnology and Biological Sciences Research Council
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