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DNA origami signposts for identifying proteins on cell membranes by electron cryotomography
DOI:
10.1016/j.cell.2021.01.033
Authors:
Emma
Silvester
(Wellcome Centre for Human Genetics, University of Oxford; Leibniz-Institut für Experimentelle Virologie)
,
Benjamin
Vollmer
(Wellcome Centre for Human Genetics, University of Oxford; Leibniz-Institut für Experimentelle Virologie)
,
Vojtech
Prazak
(Wellcome Centre for Human Genetics, University of Oxford; Leibniz-Institut für Experimentelle Virologie)
,
Daven
Vasishtan
(Wellcome Centre for Human Genetics, University of Oxford; Leibniz-Institut für Experimentelle Virologie)
,
Emily A.
Machala
(Wellcome Centre for Human Genetics, University of Oxford)
,
Catheryne
Whittle
(Wellcome Centre for Human Genetics, University of Oxford)
,
Susan
Black
(Wellcome Centre for Human Genetics, University of Oxford; Leibniz-Institut für Experimentelle Virologie)
,
Jonathan
Bath
(University of Oxford)
,
Andrew J.
Turberfield
(University of Oxford)
,
Kay
Grunewald
(Wellcome Centre for Human Genetics, University of Oxford; Leibniz-Institut für Experimentelle Virologie; University of Hamburg)
,
Lindsay A.
Baker
(Wellcome Centre for Human Genetics, University of Oxford; Leibniz-Institut für Experimentelle Virologie)
Co-authored by industrial partner:
No
Type:
Journal Paper
Journal:
Cell
, VOL 184
, PAGES 1110 - 1121.e16
State:
Published (Approved)
Published:
February 2021
Diamond Proposal Number(s):
20223

Abstract: Electron cryotomography (cryoET), an electron cryomicroscopy (cryoEM) modality, has changed our understanding of biological function by revealing the native molecular details of membranes, viruses, and cells. However, identification of individual molecules within tomograms from cryoET is challenging because of sample crowding and low signal-to-noise ratios. Here, we present a tagging strategy for cryoET that precisely identifies individual protein complexes in tomograms without relying on metal clusters. Our method makes use of DNA origami to produce “molecular signposts” that target molecules of interest, here via fluorescent fusion proteins, providing a platform generally applicable to biological surfaces. We demonstrate the specificity of signpost origami tags (SPOTs) in vitro as well as their suitability for cryoET of membrane vesicles, enveloped viruses, and the exterior of intact mammalian cells.
Journal Keywords: aptamers; cryoEM; cellular electron cryotomography; DNA origami; electron cryomicroscopy; labeling; molecular arrows; protein localisation; signpost origami tags; tagging
Subject Areas:
Biology and Bio-materials,
Technique Development
Diamond Offline Facilities:
Electron Bio-Imaging Centre (eBIC)
Instruments:
Krios IV-Titan Krios IV at Diamond
Added On:
23/02/2021 13:29
Discipline Tags:
Technique Development - Life Sciences & Biotech
Structural biology
Life Sciences & Biotech
Technical Tags:
Imaging
Tomography
Cryo Electron Tomography (Cryo ET)