17th Structural Biology Club of the Czech Society for Structural Biology
27 September 2023, 13:00
with the following scientific talks kindly delivered by our guests
Asymmetric reconstructions of immature Tick‐borne encephalitis virus particles reveal defects caused by the assembly process
Presented by Tibor Füzik, Structural Biology, Central European Institute of Technology, Masaryk University, Brno, Czech Republic
Tick‐borne encephalitis virus (TBEV) is an enveloped virus belonging to the family Flaviviridae, which causes severe disease of the central nervous system in humans. The smooth virion surface is covered by envelope proteins (E‐protein), that aretogether with the membrane proteins (M‐protein) anchored in the virus lipid bilayer. During the viral life cycle, the immature non‐infectious virus undergoes a maturation process. This process includes proteolytic cleavage of prM and a major reorganization of the envelope proteins on the viral surface. To determine the structure of immature TBEV particles, we purified them from infected tissue culture cells and used cryo‐electron microscopy for visualization. The immature particles have “spiky” surface formed by the E‐protein‐prM‐protein complex. We performed single‐particle analysis and cryo‐electron tomography to reveal the asymmetric nature of the TBEV immature particles. The symmetric, icosahedral, organization of the E‐protein‐prM‐protein spikes on the particle surface is often disrupted by defects introduced during the assembly process of the immature particle. However, these irregularities do not hinder the subsequent maturation process and instead result in mature particles with empty patches in the “herring bone” organization of the mature viral surface. The results provide further insight into the viral maturation process which could be targeted in the future by specific antiviral drugs.
High‐Confidence Particle Localization in CryoET Tomograms Using 3D Template Matching with Optimized Parameters
Presented by Beata Turoňová, Department of Molecular Sociology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany
Cryo‐electron tomography (CryoET) resolves individual macromolecules and their interactions inside living cells. However, the complex composition and high density of cells challenge the faithful identification of features in tomograms. In our current work, we capitalize on recent advances in electron tomography and demonstrate that 3D template matching (TM) localizes a wide range of structures inside crowded eukaryotic cells with confidence 10 to 100‐fold above the noise level. We show that the performance of TM not only depends on the size, origin, and shape of the template, but also on angular increment in orientational sampling, and on tomogram voxel size (magnification), filtering and resolution. We establish a TM pipeline with systematically tuned parameters for automated, objective, and comprehensive feature identification. We achieve high‐fidelity and high‐confidence localizations of various complexes (some of them as small as ~100‐kDa), demonstrating that our approach is generic. In my presentation I will demonstrate the power of optimized TM to localize nuclear pore complexes (NPCs), vault proteins, ribosomes, proteasomes, microtubules, and lipid membranes, inside a single dataset. I will show that TM quantitatively captures the diversity of eukaryotic ribosomes in different functional states and that it can identify lowabundance and low‐density complexes with high fidelity, as exemplified by the identification of ribosome‐loaded vaults. By capturing individual molecular events inside living cells with defined statistical confidence, high‐confidence TM greatly speeds up the CryoET workflow and sets the stage for visual proteomics.
Moderator: Gabriel Demo, Central European Institute of Technology, Masaryk University
Please, join us on this Zoom link (join 5‐10 minutes before the beginning)
Meeting ID: 921 7033 3913