Klebl, DP, Monteiro, DCF, Kontziampasis, D orcid.org/0000-0002-6787-8892 et al. (5 more authors) (2020) Sample deposition onto cryo-EM grids: from sprays to jets and back. Acta Crystallographica Section D: Structural Biology, 76 (4). pp. 340-349. ISSN 2059-7983
Abstract
Despite the great strides made in the field of single-particle cryogenic electron microscopy (cryo-EM) in microscope design, direct electron detectors and new processing suites, the area of sample preparation is still far from ideal. Traditionally, sample preparation involves blotting, which has been used to achieve high resolution, particularly for well behaved samples such as apoferritin. However, this approach is flawed since the blotting process can have adverse effects on some proteins and protein complexes, and the long blot time increases exposure to the damaging air–water interface. To overcome these problems, new blotless approaches have been designed for the direct deposition of the sample on the grid. Here, different methods of producing droplets for sample deposition are compared. Using gas dynamic virtual nozzles, small and high-velocity droplets were deposited on cryo-EM grids, which spread sufficiently for high-resolution cryo-EM imaging. For those wishing to pursue a similar approach, an overview is given of the current use of spray technology for cryo-EM grid preparation and areas for enhancement are pointed out. It is further shown how the broad aspects of sprayer design and operation conditions can be utilized to improve grid quality reproducibly.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2020 Klebl et al. This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
Keywords: | time-resolved; cryo-EM; sample preparation; gas dynamic virtual nozzle; structure determination; microfluidics |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Biomedical Sciences (Leeds) The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) |
Funding Information: | Funder Grant number BBSRC (Biotechnology & Biological Sciences Research Council) BB/P026397/1 |
Depositing User: | Symplectic Publications |
Date Deposited: | 04 Mar 2020 13:54 |
Last Modified: | 15 Apr 2020 15:06 |
Status: | Published |
Publisher: | International Union of Crystallography |
Identification Number: | 10.1107/S2059798320002958 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:157991 |