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