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Instruments / Technical Area	Publication Details	Published
Krios IV-Titan Krios IV at Diamond	DNA origami signposts for identifying proteins on cell membranes by electron cryotomography Emma Silvester , Benjamin Vollmer , Vojtech Prazak , Daven Vasishtan , Emily A. Machala , Catheryne Whittle , Susan Black , Jonathan Bath , Andrew J. Turberfield , Kay Grunewald , Lindsay A. Baker Cell 184, 1110 - 1121.e16 DOI: 10.1016/j.cell.2021.01.033 Diamond Proposal Number(s): [20223] Open Access Show Abstract ▼ 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.	Feb 2021
Krios I-Titan Krios I at Diamond	The prefusion structure of herpes simplex virus glycoprotein B B. Vollmer , V. Prazak , D. Vasishtan , E. E. Jefferys , A. Hernandez-Duran , M. Vallbracht , B. G. Klupp , T. C. Mettenleiter , M. Backovic , F. A. Rey , M. Topf , K. Grunewald Science Advances 6 DOI: 10.1126/sciadv.abc1726 Open Access Show Abstract ▼ Abstract: Cell entry of enveloped viruses requires specialized viral proteins that mediate fusion with the host membrane by substantial structural rearrangements from a metastable pre- to a stable postfusion conformation. This metastability renders the herpes simplex virus 1 (HSV-1) fusion glycoprotein B (gB) highly unstable such that it readily converts into the postfusion form, thereby precluding structural elucidation of the pharmacologically relevant prefusion conformation. By identification of conserved sequence signatures and molecular dynamics simulations, we devised a mutation that stabilized this form. Functionally locking gB allowed the structural determination of its membrane-embedded prefusion conformation at sub-nanometer resolution and enabled the unambiguous fit of all ectodomains. The resulting pseudo-atomic model reveals a notable conservation of conformational domain rearrangements during fusion between HSV-1 gB and the vesicular stomatitis virus glycoprotein G, despite their very distant phylogeny. In combination with our comparative sequence-structure analysis, these findings suggest common fusogenic domain rearrangements in all class III viral fusion proteins.	Sep 2020
I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Structure and mechanism of bactericidal mammalian perforin-2, an ancient agent of innate immunity Tao Ni , Fang Jiao , Xiulian Yu , Saša Aden , Lucy Ginger , Sophie I. Williams , Fangfang Bai , Vojtech Prazak , Dimple Karia , Phillip Stansfeld , Peijun Zhang , George Munson , Gregor Anderluh , Simon Scheuring , Robert J. C. Gilbert Science Advances 6 DOI: 10.1126/sciadv.aax8286 Open Access Show Abstract ▼ Abstract: Perforin-2 (MPEG1) is thought to enable the killing of invading microbes engulfed by macrophages and other phagocytes, forming pores in their membranes. Loss of perforin-2 renders individual phagocytes and whole organisms significantly more susceptible to bacterial pathogens. Here, we reveal the mechanism of perforin-2 activation and activity using atomic structures of pre-pore and pore assemblies, high-speed atomic force microscopy, and functional assays. Perforin-2 forms a pre-pore assembly in which its pore-forming domain points in the opposite direction to its membrane-targeting domain. Acidification then triggers pore formation, via a 180° conformational change. This novel and unexpected mechanism prevents premature bactericidal attack and may have played a key role in the evolution of all perforin family proteins.	Jan 2020

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