Complementing crystallography: the role of cryo-electron microscopy in structural biology (original) (raw)
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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.
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.
Protein structure determination by electron cryo-microscopy
Current Opinion in Pharmacology, 2009
Transmission electron cryo-microscopy (cryoEM) is a versatile tool in the structural analysis of proteins and biological macromolecular assemblies. In this review, we present a brief survey of the methods used in cryoEM, and their current developments. These latest advances provide exciting opportunities for the three-dimensional structural determination of macromolecular complexes that are either too large or too heterogeneous to be investigated by conventional X-ray crystallography or nuclear magnetic resonance (NMR). The endeavour of understanding the function of protein or macromolecular complex is often helped by combining data from electron microscopy and X-ray crystallography. We will thus provide a brief overview of the computational techniques involved in combining data from different techniques for the interpretation of the EM structure.
Journal of structural biology, 2017
The recent technological advances in electron microscopes, detectors, as well as image processing and reconstruction software have brought single particle cryo-electron microscopy (cryo-EM) into prominence for determining structures of bio-molecules at near atomic resolution. This has been particularly true for virus capsids, ribosomes, and other large assemblies, which have been the ideal specimens for structural studies by cryo-EM approaches. An analysis of time series metadata of virus structures on the methods of structure determination, resolution of the structures, and size of the virus particles revealed a rapid increase in the virus structures determined by cryo-EM at near atomic resolution since 2010. In addition, the data highlight the median resolution (∼3.0 Å) and size (∼310.0 Å in diameter) of the virus particles determined by X-ray crystallography while no such limits exist for cryo-EM structures, which have a median diameter of 508 Å. Notably, cryo-EM virus structures...
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...
Preservation of high resolution protein structure by cryo-electron microscopy of vitreous sections
Ultramicroscopy, 2009
We have quantitated the degree of structural preservation in cryo-sections of a vitrified biological specimen. Previous studies have used sections of periodic specimens to assess the resolution present, but preservation before sectioning was not assessed and so the damage due particularly to cutting was not clear. In this study large single crystals of lysozyme were vitrified and from these X-ray diffraction patterns extending to better than 2.1 Å were obtained. The crystals were high pressure frozen in 30% dextran, and cryo-sectioned using a diamond knife. In the best case, preservation to a resolution of 7.9 Å was shown by electron diffraction, the first observation of sub-nanometre structural preservation in a vitreous section.
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...
Resolving the geometry of biomolecules imaged by cryo electron tomography
Journal of Microscopy, 2007
In this paper, we describe two methods for computerized analysis of cryo electron tomography reconstructions of biomolecules. Both methods aim at quantifying the degree of structural flexibility of macromolecules and eventually resolving the inner dynamics through automatized protocols. The first method performs a Brownian dynamics evolution of a simplified molecular model into a fictitious force field generated by the tomograms. This procedure enables us to dock the simplified model into the experimental profiles. The second uses a fuzzy framework to delineate the subparts of the proteins and subsequently determine their interdomain relations. The two methods are discussed and their complementarities highlighted with reference to the case of the immonoglobulin antibody. Both artificial maps, constructed from immunoglobulin G entries in the Protein Data Bank and real tomograms are analyzed. Robustness issues and agreement with previously reported measurements are discussed.
Electron Cryomicroscopy of Viruses at Near-Atomic Resolutions
Annual Review of Virology, 2017
Recently, dozens of virus structures have been solved to resolutions between 2.5 and 5.0 Å by means of electron cryomicroscopy. With these structures we are now firmly within the “atomic age” of electron cryomicroscopy, as these studies can reveal atomic details of protein and nucleic acid topology and interactions between specific residues. This improvement in resolution has been the result of direct electron detectors and image processing advances. Although enforcing symmetry facilitates reaching near-atomic resolution with fewer particle images, it unfortunately obscures some biologically interesting components of a virus. New approaches on relaxing symmetry and exploring structure dynamics and heterogeneity of viral assemblies have revealed important insights into genome packaging, virion assembly, cell entry, and other stages of the viral life cycle. In the future, novel methods will be required to reveal yet-unknown structural conformations of viruses, relevant to their biolog...
Retrovirus envelope protein complex structure in situ studied by cryo-electron tomography
Proceedings of the National Academy of Sciences, 2005
We used cryo-electron tomography in conjunction with single-particle averaging techniques to study the structures of frozen-hydrated envelope glycoprotein (Env) complexes on intact Moloney murine leukemia retrovirus particles. Cryo-electron tomography allows 3D imaging of viruses in toto at a resolution sufficient to locate individual macromolecules, and local averaging of abundant complexes substantially improves the resolution. The averaging of repetitive features in electron tomograms is hampered by a low signal-to-noise ratio and anisotropic resolution, which results from the “missing-wedge” effect. We developed an iterative 3D averaging algorithm that compensates for this effect and used it to determine the trimeric structure of Env to a resolution of 2.7 nm, at which individual domains can be resolved. Strikingly, the 3D reconstruction is shaped like a tripod in which the trimer penetrates the membrane at three distinct locations ≈4.5 nm apart from one another. The Env reconst...