Conformational states of macromolecular assemblies explored by integrative structure calculation

A detailed description of macromolecular assemblies in multiple conformational states can be very valuable for understanding cellular processes. At present, structural determination of most assemblies in different biologically relevant conformations cannot be achieved by a single technique and thus requires an integrative approach that combines information from multiple sources. Different techniques require different computational methods to allow efficient and accurate data processing and analysis. Here, we summarize the latest advances and future challenges in computational methods that help the interpretation of data from two techniques—mass spectrometry and three-dimensional cryo-electron microscopy (with focus on alignment and classification of heterogeneous subtomograms from cryo-electron tomography). We evaluate how new developments in these two broad fields will lead to further integration with atomic structures to broaden our picture of the dynamic behavior of assemblies in their native environment

A convolutional autoencoder approach for mining features in cellular electron cryo-tomograms and weakly supervised coarse segmentation

Cellular electron cryo-tomography enables the 3D visualization of cellular organization in the near-native state and at submolecular resolution. However, the contents of cellular tomograms are often complex, making it difficult to automatically isolate different in situ cellular components. In this paper, we propose a convolutional autoencoder-based unsupervised approach to provide a coarse grouping of 3D small subvolumes extracted from tomograms. We demonstrate that the autoencoder can be used for efficient and coarse characterization of features of macromolecular complexes and surfaces, such as membranes. In addition, the autoencoder can be used to detect non-cellular features related to sample preparation and data collection, such as carbon edges from the grid and tomogram boundaries. The autoencoder is also able to detect patterns that may indicate spatial interactions between cellular components. Furthermore, we demonstrate that our autoencoder can be used for weakly supervised semantic segmentation of cellular components, requiring a very small amount of manual annotation.Comment: Accepted by Journal of Structural Biolog

In situ structure determination by subtomogram averaging

Cryo-tomography and subtomogram averaging are increasingly popular techniques for structural determination of macromolecular complexes in situ. They have the potential to achieve high-resolution views of native complexes, together with the details of their location relative to interacting molecules. The subtomogram averaging (StA) pipelines are well-established, with current developments aiming to optimise each step by reducing manual intervention and user decisions, following similar trends in single-particle approaches that have dramatically increased their popularity. Here, we review the main steps of typical StA workflows. We focus on considerations arising from the fact that the objects of study are embedded within unique crowded environments, and we emphasise those steps where careful decisions need to be made by the user. [Abstract copyright: Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.

In Situ Cryo-Electron Tomography: A Post-Reductionist Approach to Structural Biology

Cryo-electron tomography is a powerful technique that can faithfully image the native cellular environment at nanometer resolution. Unlike many other imaging approaches, cryo-electron tomography provides a label-free method of detecting biological structures, relying on the intrinsic contrast of frozen cellular material for direct identification of macromolecules. Recent advances in sample preparation, detector technology, and phase plate imaging have enabled the structural characterization of protein complexes within intact cells. Here, we review these technical developments and outline a detailed computational workflow for in situ structural analysis. Two recent studies are described to illustrate how this workflow can be adapted to examine both known and unknown cellular complexes. The stage is now set to realize the promise of visual proteomics a complete structural description of the cell's native molecular landscape. (C) 2015 Elsevier Ltd. All rights reserved

Structural characterization of Ebola virus uncoating

Viruses initiate infection of host cells by entering through a variety of different pathways. Their entry is concluded by the release of the viral genome into the cytoplasm, where the cellular machinery gets repurposed for virus replication. Prerequisite for genome release is the uncoating of the viral particles, a process which requires the destabilization of interactions established during virus assembly. Ebola viruses (EBOVs) are highly pathogenic, enveloped RNA viruses of remarkable filamentous morphology. Their shape is dictated by the viral matrix protein VP40, which forms a tubular scaffold underneath the viral envelope and confers stability to the particles during EBOV transmission. EBOVs enter host cells via the endocytic pathway and release their genome into the cytoplasm after fusion of their envelope with the endosomal membrane. The first line of defence against a viral infection is blocking viral entry, and EBOV entry has accordingly been well investigated with respect to receptor engagement and potential membrane fusion triggers. However, key mechanisms governing the final step of virus entry are still unknown, including the central question of how these unusually shaped virions undergo uncoating. Whether and how the VP40 matrix disassembles to enable membrane fusion; whether uncoating involves additional triggers; and finally, how and where the viral genome gets released from the viral particles and nucleocapsids remains to be elucidated. In this thesis, I investigate EBOV uncoating during entry into host cells and shed light on the fate of the most abundant and versatile viral protein, VP40. As a main tool, I use in situ cryo-electron tomography and provide structural insights into EBOV uncoating both in vitro and in infected host cells at molecular resolution. I discover that at low endosomal pH, the VP40 matrix detaches from the viral envelope and disassembles. This is caused by the disruption of electrostatic interactions between membrane lipids and anionic amino acids exposed on the surface of VP40 dimers, which I show are the structural units of the VP40 matrix. The strong effect of low pH on the integrity of the VP40 matrix is a consequence of acidification of the viral lumen, which I further investigate to uncover its mechanism. I show that protons diffuse passively across the viral envelope independently of a dedicated ion channel, which might be relevant for other late-penetrating viruses lacking viroporins. Finally, I provide the first high-resolution images of Ebola virions in endolysosomal compartments of infected cells. These images confirm the disassembly of the VP40 matrix in virions located in acidified compartments while clearly showing that their nucleocapsids remain intact. Together, these findings reveal that VP40 matrix disassembly is an essential step during EBOV uncoating, which precedes membrane fusion and genome release from the nucleocapsids. Overall, this thesis extends the current understanding of virus uncoating and indicates that pH-driven structural remodeling of viral matrix proteins may act as a switch coupling matrix uncoating to membrane fusion during host cell entry of enveloped viruses