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)