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A 3D-printed flow-cell for on-grid purification of electron microscopy samples directly from lysate
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1-s2.0-S104784772300062X-main.pdf | Published version | 10.86 MB | Adobe PDF | View/Open |
Title: | A 3D-printed flow-cell for on-grid purification of electron microscopy samples directly from lysate |
Authors: | Ramlaul, K Feng, Z Canavan, C De Martin Garrido, N Carreno, D Crone, M Jensen, K Li, B Barnett, H Riglar, D Freemont, P Miller, D Aylett, C |
Item Type: | Journal Article |
Abstract: | While recent advances in cryo-EM, coupled with single particle analysis, have the potential to allow structure determination in a near-native state from vanishingly few individual particles, this vision has yet to be realised in practise. Requirements for particle numbers that currently far exceed the theoretical lower limits, challenges with the practicalities of achieving high concentrations for difficult-to-produce samples, and inadequate sample-dependent imaging conditions, all result in significant bottlenecks preventing routine structure determination using cryo-EM. Therefore, considerable efforts are being made to circumvent these bottlenecks by developing affinity purification of samples on-grid; at once obviating the need to produce large amounts of protein, as well as more directly controlling the variable, and sample-dependent, process of grid preparation. In this proof-of-concept study, we demonstrate a further practical step towards this paradigm, developing a 3D-printable flow-cell device to allow on-grid affinity purification from raw inputs such as whole cell lysates, using graphene oxide-based affinity grids. Our flow-cell device can be interfaced directly with routinely-used laboratory equipment such as liquid chromatographs, or peristaltic pumps, fitted with standard chromatographic (1/16”) connectors, and can be used to allow binding of samples to affinity grids in a controlled environment prior to the extensive washing required to remove impurities. Furthermore, by designing a device which can be 3D printed and coupled to routinely used laboratory equipment, we hope to increase the accessibility of the techniques presented herein to researchers working towards single-particle macromolecular structures. |
Issue Date: | Sep-2023 |
Date of Acceptance: | 10-Jul-2023 |
URI: | http://hdl.handle.net/10044/1/105375 |
DOI: | 10.1016/j.jsb.2023.107999 |
ISSN: | 1047-8477 |
Publisher: | Elsevier |
Start Page: | 1 |
End Page: | 12 |
Journal / Book Title: | Journal of Structural Biology |
Volume: | 215 |
Issue: | 3 |
Copyright Statement: | © 2023 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
Publication Status: | Published |
Article Number: | 107999 |
Online Publication Date: | 2023-07-13 |
Appears in Collections: | Department of Infectious Diseases Faculty of Medicine |
This item is licensed under a Creative Commons License