Protein structure determination by electron cryo-microscopy (original) (raw)
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Microscopy (Oxford, England), 2015
Single particle cryo-EM has recently developed into a powerful tool to determine the 3D structure of macromolecular complexes at near-atomic resolution, which allows structural biologists to build atomic models of proteins. All technical aspects of cryo-EM technology have been considerably improved over the last two decades, including electron microscopic hardware, image processing software and the ever growing speed of computers. This leads to a more widespread use of the technique, and it can be anticipated that further automation of electron microscopes and image processing tools will soon fully shift the focus away from the technological aspects, onto biological questions that can be answered. In single particle cryo-EM, no crystals of a macromolecule are required. In contrast to X-ray crystallography, this significantly facilitates structure determination by cryo-EM. Nevertheless, a relatively high level of biochemical control is still essential to obtain high-resolution struct...
iScience, 2021
Summary Cellular factories engage numerous highly complex “molecular machines” to perform pivotal biological functions. 3D structural visualization is an effective way to understand the functional mechanisms of these biomacromolecules. The “resolution revolution” has established cryogenic electron microscopy (cryo-EM) as a preferred structural biology tool. In parallel with the advances in cryo-EM methodologies aiming at atomic resolution, several innovative approaches have started emerging where other techniques are sensibly integrated with cryo-EM to obtain additional insights into the biological processes. For example, combining the time-resolved technique with high-resolution cryo-EM enables discerning structures of short-lived intermediates in the functional pathway of a biomolecule. Likewise, integrating mass spectrometry (MS) techniques with cryo-EM allows deciphering structural organizations of large molecular assemblies. Here, we discuss how the data generated upon combinin...
Complementing crystallography: the role of cryo-electron microscopy in structural biology
Acta Crystallographica Section D Biological Crystallography, 1999
Dramatic improvements in experimental methods and computational techniques have revolutionized three-dimensional image reconstruction from electron micrographs (EM) of vitri®ed samples. Recent results include the ®rst determination of a protein fold (for the core protein of the hepatitis B virus) by non-crystalline imaging techniques. These developments have generated interest within the crystallographic community and have led to a re-evaluation of the technique, particularly amongst those working in the ®eld of virus structure or struggling with the phasing of large macromolecular assemblies. A simple discussion of the techniques of EM image reconstruction and its advantages and problems in terms familiar to crystallographers will hopefully allow an appreciation of the essential complementarity of the two techniques and the practical potentials for phasing applications.
2005
We explore structural characterization of protein assemblies by a combination of electron cryo-microscopy (cryoEM) and comparative protein structure modeling. Specifically, our method finds an optimal atomic model of a given assembly subunit and its position within an assembly by fitting alternative comparative models into a cryoEM map. The alternative models are calculated by MODELLER [J. Mol. Biol. 234 (1993) 313] from different sequence alignments between the modeled protein and its template structures.
Three-dimensional electron cryo-microscopy as a powerful structural tool in molecular medicine
Electron cryo-microscopy has established itself as a valuable method for the structure determination of protein molecules, protein complexes, and cell organelles. This contribution presents an introduction to the various aspects of three-dimensional electron cryomicroscopy. This includes the need for sample preservation in the microscope vacuum, strategies for minimizing radiation damage, methods of improving the poor signalto-noise ratio in electron micrographs of unstained specimens, and the various methods of three-dimensional image reconstruction from projections. The various specimen types (e.g., flat and tubular two-dimensional crystals, protein filaments, individual protein molecules, and large complexes) require different means of three-dimensional reconstruction, and we review the five major reconstruction techniques (electron crystallography, heli-cal reconstruction, icosahedral reconstruction, singleparticle reconstruction, and electron tomography), with an emphasis on electron crystallography. Several medically relevant three-dimensional protein structures are chosen to illustrate the potential of electron cryo-microscopy and image reconstruction techniques. Among the structural methods, electron cryo-microscopy is the only tool for studying objects that range in size from small proteins over macromolecular complexes to cell organelles or even cells.
Abstract Single particle reconstruction using Cryo-Electron Microscopy (cryo-EM) is an emerging technique in structural biology for estimating the 3-D structure (density) of protein macromolecules. Unlike tomography where a large number of images of a specimen can be acquired, the number of images of an individual particle is limited because of radiation damage. Instead, the specimen consists of identical copies of the same protein macro-molecule embedded in vitreous ice at random and unknown 3-D orientations.
The integrative role of cryo electron microscopy in molecular and cellular structural biology
Biology of the cell, 2017
After gradually moving away from preparation methods prone to artefacts such as plastic embedding and negative staining for cell sections and single particles, the field of cryo electron microscopy (cryo-EM) is now heading off at unprecedented speed towards high-resolution analysis of biological objects of various sizes. This 'revolution in resolution' is happening largely thanks to new developments of new-generation cameras used for recording the images in the cryo electron microscope which have much increased sensitivity being based on complementary metal oxide semiconductor devices. Combined with advanced image processing and 3D reconstruction, the cryo-EM analysis of nucleoprotein complexes can provide unprecedented insights at molecular and atomic levels and address regulatory mechanisms in the cell. These advances reinforce the integrative role of cryo-EM in synergy with other methods such as X-ray crystallography, fluorescence imaging or focussed-ion beam milling as e...
Template-free detection of macromolecular complexes in cryo electron tomograms
Bioinformatics, 2011
Motivation: Cryo electron tomography (CryoET) produces 3D density maps of biological specimen in its near native states. Applied to small cells, cryoET produces 3D snapshots of the cellular distributions of large complexes. However, retrieving this information is non-trivial due to the low resolution and low signal-to-noise ratio in tomograms. Current pattern recognition methods identify complexes by matching known structures to the cryo electron tomogram. However, so far only a small fraction of all protein complexes have been structurally resolved. It is, therefore, of great importance to develop template-free methods for the discovery of previously unknown protein complexes in cryo electron tomograms. Results: Here, we have developed an inference method for the template-free discovery of frequently occurring protein complexes in cryo electron tomograms. We provide a first proof-of-principle of the approach and assess its applicability using realistically simulated tomograms, allo...