Project

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Upgrade of a Titan Krios electron cryo-microscope for single particle analysis and tomography

Applicant Pilhofer Martin
Number 164092
Funding scheme R'EQUIP
Research institution Institut für Molekularbiologie und Biophysik Deptartement für Biologie ETH Zurich
Institution of higher education ETH Zurich - ETHZ
Main discipline Biophysics
Start/End 01.12.2015 - 31.01.2018
Approved amount 1'000'000.00
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All Disciplines (6)

Discipline
Biophysics
Biochemistry
Cellular Biology, Cytology
Structural Research
Molecular Biology
Experimental Microbiology

Keywords (10)

Titan Krios; Phaseplate; Single Particle Analysis; Direct Electron Detector; Structural Biology; Cell Biology; Electron Cryo-Tomography; Imaging Filter; Imaging Across Scales; Electron Cryo-Microscopy

Lay Summary (German)

Lead
Die Visualisierung von zellulären Komponenten ist der leistungsfähigste Ansatz die Zellfunktion zu verstehen. Dazu hat sich die Elektronen Kryomikroskopie als eine der am am besten geeigneten Methoden herausgestellt. Insbesondere die Modalitäten der Einzelpartikelanalyse und die Tomographie haben kürzlich zu einer Revolution in der Struktur- und Zellbiologie geführt.
Lay summary

An der ETH Zürich steht in der Imaging-Einrichtung ScopeM ein hochmodernes Titan Krios Elektronen Kryomikroskop zur Verfügung. Dieses ist jedoch technisch limitiert auf Einzelpartikelanalyse von großen Partikeln. Einzelpartikelanalysen von kleinen Partikeln sowie Tomographie können derzeit nicht kompetitiv durchgeführt werden. Dies ist in einer der Hauptlimitierungen der Elektronen Kryomikroskopie begründet: dem “Signal-to-noise” Verhältnis.

 

In diesem Projekt schlagen wir vor, das Signal-to-noise Verhältnis und damit die Leitungsfähigkeit des Titan Krios durch eine Reihe von Upgrades deutlich zu verbessern. Dies wird dazu führen, das volle Potenzial des Titan Krios ausnutzen zu können und den Kreis der Benutzer zu erweitern.

Direct link to Lay Summary Last update: 16.11.2015

Responsible applicant and co-applicants

Publications

Publication
Structure of a eukaryotic cytoplasmic pre‐40S ribosomal subunit
Scaiola Alain, Peña Cohue, Weisser Melanie, Böhringer Daniel, Leibundgut Marc, Klingauf‐Nerurkar Purnima, Gerhardy Stefan, Panse Vikram Govind, Ban Nenad (2018), Structure of a eukaryotic cytoplasmic pre‐40S ribosomal subunit, in EMBO Journal, e98499.
Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation.
Wild Rebekka, Kowal Julia, Eyring Jillianne, Ngwa Elsy M, Aebi Markus, Locher Kaspar P (2018), Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation., in Science (New York, N.Y.), xx.
In Situ Imaging of Bacterial Secretion Systems by Electron Cryotomography.
Weiss Gregor L, Medeiros João M, Pilhofer Martin (2017), In Situ Imaging of Bacterial Secretion Systems by Electron Cryotomography., in Methods in molecular biology (Clifton, N.J.), 1615, 353-375.
Prophage-triggered membrane vesicle formation through peptidoglycan damage in Bacillus subtilis.
Toyofuku Masanori, Cárcamo-Oyarce Gerardo, Yamamoto Tatsuya, Eisenstein Fabian, Hsiao Chien-Chi, Kurosawa Masaharu, Gademann Karl, Pilhofer Martin, Nomura Nobuhiko, Eberl Leo (2017), Prophage-triggered membrane vesicle formation through peptidoglycan damage in Bacillus subtilis., in Nature communications, 8(1), 481-481.
In situ architecture, function, and evolution of a contractile injection system.
Böck Désirée, Medeiros João M, Tsao Han-Fei, Penz Thomas, Weiss Gregor L, Aistleitner Karin, Horn Matthias, Pilhofer Martin (2017), In situ architecture, function, and evolution of a contractile injection system., in Science (New York, N.Y.), 357(6352), 713-717.

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
FEMS Meeting 2017 Talk given at a conference Structure, function, and evolution of a bacterial contractile injection system 09.07.2017 Valencia, Spain Pilhofer Martin;
Gordon 3DEM Talk given at a conference Structure, function, and evolution of bacterial contractile injection systems 11.06.2017 Les Diablerets, Switzerland Pilhofer Martin;


Communication with the public

Communication Title Media Place Year
Media relations: print media, online media Bacteria stab amoebae with daggers International 2017
Media relations: radio, television Tagesschau SRF German-speaking Switzerland 2017

Awards

Title Year
Ernst Jung Prize for Medicine 2017

Associated projects

Number Title Start Funding scheme
144214 Structural studies of eukaryotic complexes involved in ribosome assembly and translation initiation 01.11.2012 Project funding (Div. I-III)
152878 Structure and Mechanism of Bacterial Contractile Assemblies 01.12.2014 Project funding (Div. I-III)
141735 NCCR RNA & disease: Understanding the role of RNA biology in disease mechanisms (phase I) 01.05.2014 National Centres of Competence in Research (NCCRs)
160312 Function and regulation of cullin-based E3 ubiquitin ligases 01.04.2015 Project funding (Div. I-III)
179255 Structure, function, and evolution of bacterial contractile injection systems 01.04.2018 Project funding (Div. I-III)
144035 In vivo and in vitro structural analysis of flagellar/ciliary motors and regulators 01.07.2013 Project funding (Div. I-III)
125762 NCCR TransCure: From transport physiology to identification of therapeutic targets (phase I) 01.11.2010 National Centres of Competence in Research (NCCRs)
147632 Transglyco: Chemistry, Enzymology and Physiology of Oligosaccharyltransferase 01.11.2013 Sinergia
150665 Structural molecular biology of membrane proteins in signal transduction and cholesterol recognition 01.04.2014 SNSF Professorships
154421 ER-phagy mechanisms to maintain and restore endoplasmic reticulum homeostasis 01.10.2014 Sinergia
146191 Reaction mechanism of bacterial ABC transporters and oligosaccharyltransferase 01.04.2013 Project funding (Div. I-III)
146284 Molecular and structural characterization of Listeria bacteriophage-host interactions: from protein function to supermolecular structure 01.10.2013 Project funding (Div. I-III)
192644 Structural study on calcium regulation of motile cilia by cryo-EM 01.04.2020 Project funding (Div. I-III)
170808 Acquisition of a Talos Arctica transmission electron microscope for single particle analysis and cryo-tomography 01.05.2017 R'EQUIP
155823 Rewiring the DNA repair machinery for genome stability and haploidisation 01.04.2015 SNSF Starting Grants
200294 Systematic cryo-EM and proteomic analysis of protein complexes related to primary ciliary dyskinesia 01.11.2021 India

Abstract

Visualizing structures of cellular components is the most powerful approach to understand cellular function. In recent years, transmission electron microscopy of frozen-hydrated specimen (electron cryo-microscopy, cryoEM) has emerged as one of the most powerful methods for these studies. Two cryoEM modalities, single particle analysis and electron cryo-tomography (ECT), deliver highly complementary data. In single particle cryoEM, projection images of thousands of individual copies of the assembly are averaged and then used to calculate a 3D structure. Since very recently, this approach is able to resolve the structure of purified asymmetric macromolecular complexes at atomic resolution, with significant advantages over X-ray crystallography. In ECT, the target is imaged by a series of projections from different angles, which are reconstructed into a 3D-image (tomogram). This allows for structure determination of unique objects such as cells or cell organelles. Importantly, the structure of macromolecular complexes can be solved in the cellular context (in situ) and in the nanometer range of resolution. ECT is thus considered a bridging technology between Structural and Cell Biology.The poor contrast and signal-to-noise-ratio (SNR) obtained from radiation-sensitive biological samples presents the major limitation in cryoEM. Recent instrument developments have thus aimed at improving contrast and consequently SNR. While the imaging platform (ScopeM) at ETH Zürich operates a state-of-the-art 300 keV electron cryo-microscope (FEI Titan Krios), the current instrument setup has significant limitations that are three-fold: 1) Superior detectors are available for single particle analysis of megadalton-sized complexes. 2) Shortcomings of the current detector do not allow for single particle structure determination of small or featureless assemblies. 3) The lack of an imaging filter does not allow for cellular ECT.Here, we propose to significantly enhance the performance (contrast/SNR) of the Titan Krios microscope for single particle and ECT investigations. The proposed package includes an upgrade of the Falcon direct electron detector from version II to III, the installation of an imaging filter/K2XP Summit direct electron detector bundle, and the installation of a phase plate. The upgrade will enhance the usability of the Titan Krios instrument by 1) improving efficiency and data quality for single particle cryoEM of large assemblies, 2) enabling single particle cryoEM of small particles, and 3) enabling state-of-the-art ECT of cells and organelles. Consequently, the upgrade will facilitate the possibility to use the full scientific potential of the Titan Krios and at the same time expand the circle of users. Our group of applicants from the ETH Zürich Department of Biology and the Paul Scherrer Institute will benefit from the improvements stated above to investigate the biology of ribosomes (Ban), eukaryotic flagella (Ishikawa), adenylyl cyclases (Korkhov), ABC transporters (Locher), meiotic chromosomes (Matos), cullin-RING E3 ligases (Peter), and bacterial cell-cell interactions (Pilhofer). The majority of the proposed projects are not feasible with the current instrumentation. The upgraded setup will also foster interdisciplinary approaches to image across scales, being a scientific focus area of the Department of Biology at ETH Zürich.
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