scholarly journals Introduction to high-resolution cryo-electron microscopy

2016 ◽  
Vol 62 (3) ◽  
pp. 383-394
Author(s):  
Mariusz Czarnocki-Cieciura ◽  
Marcin Nowotny
Keyword(s):  
Electron Microscopy ◽  
Nmr Spectroscopy ◽  
Structural Biology ◽  
Atomic Resolution ◽  
Biological Macromolecules ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Electron Microscopes

For many years two techniques have dominated structural biology – X-ray crystallography and NMR spectroscopy. Traditional cryo-electron microscopy of biological macromolecules produced macromolecular reconstructions at resolution limited to 6–10 Å. Recent development of transmission electron microscopes, in particular the development of direct electron detectors, and continuous improvements in the available software, have led to the “resolution revolution” in cryo-EM. It is now possible to routinely obtain near-atomic-resolution 3D maps of intact biological macromolecules as small as ~100 kDa. Thus, cryo-EM is now becoming the method of choice for structural analysis of many complex assemblies that are unsuitable for structure determination by other methods.

scholarly journals Visualization of biological macromolecules at near-atomic resolution: cryo-electron microscopy comes of age

2019 ◽  
Vol 75 (1) ◽  
pp. 3-11 ◽  
Author(s):  
Alok K. Mitra
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Atomic Resolution ◽  
Particle Analysis ◽  
Biological Macromolecules ◽  
Single Particles ◽  
Cryo Electron Microscopy ◽  
Electron Microscopes ◽  
Resolution Single ◽  
Resolution Structure

Structural biology is going through a revolution as a result of transformational advances in the field of cryo-electron microscopy (cryo-EM) driven by the development of direct electron detectors and ultrastable electron microscopes. High-resolution cryo-EM images of isolated biomolecules (single particles) suspended in a thin layer of vitrified buffer are subjected to powerful image-processing algorithms, enabling near-atomic resolution structures to be determined in unprecedented numbers. Prior to these advances, electron crystallography of two-dimensional crystals and helical assemblies of proteins had established the feasibility of atomic resolution structure determination using cryo-EM. Atomic resolution single-particle analysis, without the need for crystals, now promises to resolve problems in structural biology that were intractable just a few years ago.

scholarly journals Advances in methods for atomic resolution macromolecular structure determination

F1000Research ◽  
2020 ◽  
Vol 9 ◽  
pp. 667
Author(s):  
Michael C. Thompson ◽  
Todd O. Yeates ◽  
Jose A. Rodriguez
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Atomic Resolution ◽  
Macromolecular Structure ◽  
New Developments ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Technical Advances ◽  
Dynamical Properties

Recent technical advances have dramatically increased the power and scope of structural biology. New developments in high-resolution cryo-electron microscopy, serial X-ray crystallography, and electron diffraction have been especially transformative. Here we highlight some of the latest advances and current challenges at the frontiers of atomic resolution methods for elucidating the structures and dynamical properties of macromolecules and their complexes.

scholarly journals From X-ray crystallography to cryo-electron microscopy: computing infrastructure in structural biology

2018 ◽  
Vol 74 (a1) ◽  
pp. a224-a224
Author(s):  
Jason Key ◽  
Peter A. Meyer ◽  
Carol Herre ◽  
Michael Timony ◽  
Dimitry Filonov ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Computing Infrastructure

scholarly journals A basic introduction to single particles cryo-electron microscopy

2021 ◽  
Vol 9 (1) ◽  
pp. 5-20
Author(s):  
Vittoria Raimondi ◽  
◽  
Alessandro Grinzato ◽  
◽  
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Protein Complexes ◽  
Computational Power ◽  
Single Particles ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
First Choice ◽  
New Generation

<abstract> <p>In the last years, cryogenic-electron microscopy (cryo-EM) underwent the most impressive improvement compared to other techniques used in structural biology, such as X-ray crystallography and NMR. Electron microscopy was invented nearly one century ago but, up to the beginning of the last decades, the 3D maps produced through this technique were poorly detailed, justifying the term “blobbology” to appeal to cryo-EM. Recently, thanks to a new generation of microscopes and detectors, more efficient algorithms, and easier access to computational power, single particles cryo-EM can routinely produce 3D structures at resolutions comparable to those obtained with X-ray crystallography. However, unlike X-ray crystallography, which needs crystallized proteins, cryo-EM exploits purified samples in solution, allowing the study of proteins and protein complexes that are hard or even impossible to crystallize. For these reasons, single-particle cryo-EM is often the first choice of structural biologists today. Nevertheless, before starting a cryo-EM experiment, many drawbacks and limitations must be considered. Moreover, in practice, the process between the purified sample and the final structure could be trickier than initially expected. Based on these observations, this review aims to offer an overview of the principal technical aspects and setups to be considered while planning and performing a cryo-EM experiment.</p> </abstract>

scholarly journals Structure Determination by Single-Particle Cryo-Electron Microscopy: Only the Sky (and Intrinsic Disorder) is the Limit

2019 ◽  
Vol 20 (17) ◽  
pp. 4186 ◽  
Author(s):  
Emeka Nwanochie ◽  
Vladimir N. Uversky
Keyword(s):  
Electron Microscopy ◽  
Nmr Spectroscopy ◽  
Structure Determination ◽  
Single Particle ◽  
Protein Structures ◽  
X Ray ◽  
X Ray Crystallography ◽  
Intrinsically Disordered ◽  
Small Proteins ◽  
Cryo Electron Microscopy

Traditionally, X-ray crystallography and NMR spectroscopy represent major workhorses of structural biologists, with the lion share of protein structures reported in protein data bank (PDB) being generated by these powerful techniques. Despite their wide utilization in protein structure determination, these two techniques have logical limitations, with X-ray crystallography being unsuitable for the analysis of highly dynamic structures and with NMR spectroscopy being restricted to the analysis of relatively small proteins. In recent years, we have witnessed an explosive development of the techniques based on Cryo-electron microscopy (Cryo-EM) for structural characterization of biological molecules. In fact, single-particle Cryo-EM is a special niche as it is a technique of choice for the structural analysis of large, structurally heterogeneous, and dynamic complexes. Here, sub-nanometer atomic resolution can be achieved (i.e., resolution below 10 Å) via single-particle imaging of non-crystalline specimens, with accurate 3D reconstruction being generated based on the computational averaging of multiple 2D projection images of the same particle that was frozen rapidly in solution. We provide here a brief overview of single-particle Cryo-EM and show how Cryo-EM has revolutionized structural investigations of membrane proteins. We also show that the presence of intrinsically disordered or flexible regions in a target protein represents one of the major limitations of this promising technique.

scholarly journals Cryo-electron microscopy and X-ray crystallography: complementary approaches to structural biology and drug discovery

2017 ◽  
Vol 73 (4) ◽  
pp. 174-183 ◽  
Author(s):  
Catherine Vénien-Bryan ◽  
Zhuolun Li ◽  
Laurent Vuillard ◽  
Jean Albert Boutin
Keyword(s):  
Electron Microscopy ◽  
Image Analysis ◽  
High Resolution ◽  
New Drugs ◽  
Macromolecular Complexes ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Reconstruction Methods ◽  
Electron Microscopes

The invention of the electron microscope has greatly enhanced the view scientists have of small structural details. Since its implementation, this technology has undergone considerable evolution and the resolution that can be obtained for biological objects has been extended. In addition, the latest generation of cryo-electron microscopes equipped with direct electron detectors and software for the automated collection of images, in combination with the use of advanced image-analysis methods, has dramatically improved the performance of this technique in terms of resolution. While calculating a sub-10 Å resolution structure was an accomplishment less than a decade ago, it is now common to generate structures at sub-5 Å resolution and even better. It is becoming possible to relatively quickly obtain high-resolution structures of biological molecules, in particular large ones (>500 kDa) which, in some cases, have resisted more conventional methods such as X-ray crystallography or nuclear magnetic resonance (NMR). Such newly resolved structures may, for the first time, shed light on the precise mechanisms that are essential for cellular physiological processes. The ability to attain atomic resolution may support the development of new drugs that target these proteins, allowing medicinal chemists to understand the intimacy of the relationship between their molecules and targets. In addition, recent developments in cryo-electron microscopy combined with image analysis can provide unique information on the conformational variability of macromolecular complexes. Conformational flexibility of macromolecular complexes can be investigated using cryo-electron microscopy and multiconformation reconstruction methods. However, the biochemical quality of the sample remains the major bottleneck to routine cryo-electron microscopy-based determination of structures at very high resolution.

scholarly journals Achieving better than 3 Å resolution by single particle cryo-EM at 200 keV

2017 ◽  
Author(s):  
Mark A. Herzik ◽  
Mengyu Wu ◽  
Gabriel C. Lander
Keyword(s):  
Single Particle ◽  
Water Molecules ◽  
Macromolecular Complexes ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Electron Microscopes ◽  
Ordered Water ◽  
Better Than

AbstractTechnical and methodological advances in single-particle cryo-electron microscopy (cryo-EM) have expanded the technique into a resolution regime that was previously only attainable by X-ray crystallography. Although single-particle cryo-EM has proven to be a useful technique for determining the structures of biomedically relevant molecules at near-atomic resolution, nearly 98% of the structures resolved to better than 4 Å resolution have been determined using 300 keV transmission electron microscopes (TEMs). We demonstrate that it is possible to obtain cryo-EM reconstructions of macromolecular complexes at a range of sizes to better than 3 Å resolution using a 200 keV TEM. These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules, features previously thought only to be resolvable using TEMs operating at 300 keV.

scholarly journals On the explicit use of experimental images in high resolution cryo-EM refinement

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 665
Author(s):  
Jacqueline Cherfils ◽  
Jorge Navaza
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Structural Biology ◽  
Single Particle ◽  
Atomic Resolution ◽  
Full Potential ◽  
X Ray ◽  
X Ray Crystallography ◽  
Atomic Models

Single particle cryogenic electron microscopy (cryo-EM) is transforming structural biology by enabling the analysis of difficult macromolecular specimens, such as membrane proteins or large complexes with flexible elements, at near atomic resolution with an accuracy close to that of X-ray crystallography. As the technique continues to improve, it is important to assess and exploit its full potential to produce the most possible reliable atomic models. Here we propose to use the experimental images as the data for refinement and validation, instead of the reconstructed maps as currently used. This procedure, which is in spirit quite similar to that used in X-ray crystallography where the data include experimental phases, should contribute to improve the quality of the cryo-EM atomic models.

scholarly journals LA RIVOLUZIONE DELLA CRIO-MICROSCOPIA ELETTRONICA NELLO STUDIO STRUTTURALE DI PROTEINE E ACIDI NUCLEICI

2020 ◽  
Author(s):  
Martino Bolognesi
Keyword(s):  
Electron Microscopy ◽  
Amino Acids ◽  
Structural Biology ◽  
Molecular Structures ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Past ◽  
Cryo Electron Microscopy ◽  
Resolution Level ◽  
Nello Studio

Observing the fine details of molecular structures (e.g. in proteins and in nucleic acids) has been a central part of Structural Biology over the past 50 years. The recent advent of single particle cryo-electron microscopy brought a revolution in this field, that previously relied on X-ray crystallography and nuclear magnetic resonance. It is now possible to explore the structures of large subcellular assemblies, such as the ribosome, resolving details on the scale of amino acids and nucleotides, in favorable cases reaching the 2 Å resolution level.

Sign in / Sign up

Export Citation Format

Share Document