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Nature Nov 2020The three-dimensional positions of atoms in protein molecules define their structure and their roles in biological processes. The more precisely atomic coordinates are...
The three-dimensional positions of atoms in protein molecules define their structure and their roles in biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more mechanistic insights into protein function may be inferred. Electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years. However, it has proved difficult to obtain cryo-EM reconstructions with sufficient resolution to visualize individual atoms in proteins. Here we use a new electron source, energy filter and camera to obtain a 1.7 Å resolution cryo-EM reconstruction for a human membrane protein, the β3 GABA receptor homopentamer. Such maps allow a detailed understanding of small-molecule coordination, visualization of solvent molecules and alternative conformations for multiple amino acids, and unambiguous building of ordered acidic side chains and glycans. Applied to mouse apoferritin, our strategy led to a 1.22 Å resolution reconstruction that offers a genuine atomic-resolution view of a protein molecule using single-particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualized in difference maps, allowing a direct analysis of hydrogen-bonding networks. Our technological advances, combined with further approaches to accelerate data acquisition and improve sample quality, provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery.
Topics: Animals; Apoferritins; Cryoelectron Microscopy; Drug Discovery; Humans; Mice; Models, Molecular; Polysaccharides; Receptors, GABA-A; Single Molecule Imaging
PubMed: 33087931
DOI: 10.1038/s41586-020-2829-0 -
Signal Transduction and Targeted Therapy Feb 2022Cryo-electron microscopy (cryo-EM) has emerged as an unprecedented tool to resolve protein structures at atomic resolution. Structural insights of biological samples not... (Review)
Review
Cryo-electron microscopy (cryo-EM) has emerged as an unprecedented tool to resolve protein structures at atomic resolution. Structural insights of biological samples not accessible by conventional X-ray crystallography and NMR can be explored with cryo-EM because measurements are carried out under near-native crystal-free conditions, and large protein complexes with conformational and compositional heterogeneity are readily resolved. RNA has remained underexplored in cryo-EM, despite its essential role in various biological processes. This review highlights current challenges and recent progress in using cryo-EM single-particle analysis to determine protein-free RNA structures, enabled by improvement in sample preparation and integration of multiple structural and biochemical methods.
Topics: Cryoelectron Microscopy; RNA
PubMed: 35197441
DOI: 10.1038/s41392-022-00916-0 -
Structure (London, England : 1993) May 2018Recent advances in instrumentation and image-processing software have resulted in a resolution revolution in cryo-electron microscopy (cryo-EM) and a surge in the...
Recent advances in instrumentation and image-processing software have resulted in a resolution revolution in cryo-electron microscopy (cryo-EM) and a surge in the popularity of this technique. However, despite technical progress and hundreds of structures determined so far, development of standards assessing the agreement between the cryo-EM map and the respective model has fallen behind. Here we establish a validation procedure evaluating this agreement and applied it to a set of 565 cryo-EM structures. Analysis of the results revealed that three-quarters of the validated structures exhibit moderate or low agreement between the map and the corresponding model, mostly due to limited structural features possessed by these maps. Model re-refinement significantly improved the agreement for only one-fifth of the structures, reaffirming the necessity to re-evaluate map resolution. The presented procedure provides an approach to re-estimate the resolution of cryo-EM map areas interpreted by the model.
Topics: Cryoelectron Microscopy; Crystallography, X-Ray; Databases, Protein; Image Processing, Computer-Assisted; Models, Molecular; Protein Conformation; Proteins
PubMed: 29606592
DOI: 10.1016/j.str.2018.03.004 -
Current Biology : CB Oct 2018Eva Nogales introduces cryo-electron microscopy used to visualize macromolecular structures. (Review)
Review
Eva Nogales introduces cryo-electron microscopy used to visualize macromolecular structures.
Topics: Cryoelectron Microscopy; Macromolecular Substances
PubMed: 30300591
DOI: 10.1016/j.cub.2018.07.016 -
Journal of Structural Biology Dec 2018
Comparative Study
Topics: Apoferritins; Consensus Development Conferences as Topic; Cryoelectron Microscopy; Humans; Models, Molecular; Protein Conformation; TRPV Cation Channels
PubMed: 30321594
DOI: 10.1016/j.jsb.2018.10.004 -
Chemical Reviews Sep 2022While the application of cryogenic electron microscopy (cryo-EM) to helical polymers in biology has a long history, due to the huge number of helical macromolecular... (Review)
Review
While the application of cryogenic electron microscopy (cryo-EM) to helical polymers in biology has a long history, due to the huge number of helical macromolecular assemblies in viruses, bacteria, archaea, and eukaryotes, the use of cryo-EM to study synthetic soft matter noncovalent polymers has been much more limited. This has mainly been due to the lack of familiarity with cryo-EM in the materials science and chemistry communities, in contrast to the fact that cryo-EM was developed as a biological technique. Nevertheless, the relatively few structures of self-assembled peptide nanotubes and ribbons solved at near-atomic resolution by cryo-EM have demonstrated that cryo-EM should be the method of choice for a structural analysis of synthetic helical filaments. In addition, cryo-EM has also demonstrated that the self-assembly of soft matter polymers has enormous potential for polymorphism, something that may be obscured by techniques such as scattering and spectroscopy. These cryo-EM structures have revealed how far we currently are from being able to predict the structure of these polymers due to their chaotic self-assembly behavior.
Topics: Cryoelectron Microscopy; Macromolecular Substances; Polymers; Viruses
PubMed: 35133794
DOI: 10.1021/acs.chemrev.1c00753 -
Advances in Protein Chemistry and... 2011Cryo-electron microscopy (cryo-EM) in combination with single-particle analysis has begun to complement crystallography in the study of large macromolecules at... (Review)
Review
Cryo-electron microscopy (cryo-EM) in combination with single-particle analysis has begun to complement crystallography in the study of large macromolecules at near-atomic resolution. Furthermore, advances in cryo-electron tomography have made possible the study of macromolecules within their cellular environment. Single-particle and tomographic studies will become even more useful when technologies for improving the signal-to-noise ratio such as direct electron detectors and phase plates become widely available. Automated image acquisition has significantly reduced the time and effort required to determine the structures of macromolecular assemblies. As a result, the number of structures determined by cryo-EM is growing exponentially. However, there is an urgent need for improved criteria for validating both the reconstruction process and the atomic models derived from cryo-EM data. Another major challenge will be mitigating the effects of anisotropy caused by the missing wedge and the excessively low signal-to-noise ratio for tomographic data. Parallels between the development of macromolecular crystallography and cryo-EM have been used to tentatively predict the future of cryo-EM.
Topics: Animals; Cryoelectron Microscopy; Humans
PubMed: 21501821
DOI: 10.1016/B978-0-12-386507-6.00005-1 -
Current Opinion in Structural Biology Apr 2023The cryo-electron microscopy (cryo-EM) method microcrystal electron diffraction (MicroED) was initially described in 2013 and has recently gained attention as an... (Review)
Review
The cryo-electron microscopy (cryo-EM) method microcrystal electron diffraction (MicroED) was initially described in 2013 and has recently gained attention as an emerging technique for research in drug discovery. As compared to other methods in structural biology, MicroED provides many advantages deriving from the use of nanocrystalline material for the investigations. Here, we review the recent advancements in the field of MicroED and show important examples of small molecule, peptide and protein structures that has contributed to the current development of this method as an important tool for drug discovery.
Topics: Cryoelectron Microscopy; Electrons; Models, Molecular; Proteins; Drug Discovery
PubMed: 36821888
DOI: 10.1016/j.sbi.2023.102549 -
Trends in Biochemical Sciences Feb 2022Cryogenic electron microscopy (cryoEM) uses images of frozen hydrated biological specimens to produce macromolecular structures, opening up previously inaccessible... (Review)
Review
Cryogenic electron microscopy (cryoEM) uses images of frozen hydrated biological specimens to produce macromolecular structures, opening up previously inaccessible levels of biological organization to high-resolution structural analysis. CryoEM has the potential for broad impact in biomedical research, including basic cell, molecular, and structural biology, and increasingly in drug discovery and vaccine development. Recent advances have led to the expansion of molecular and cellular structure determination at an exponential rate. National and regional centers have emerged to support this growth by increasing the accessibility of cryoEM throughout the biomedical research community. Through cooperation and synergy, these centers form a network of resources that accelerate the adoption of best practices for access and training and establish sustainable workflows to build future research capacity.
Topics: Cryoelectron Microscopy; Molecular Structure
PubMed: 34823974
DOI: 10.1016/j.tibs.2021.10.007 -
Journal of Bacteriology Apr 2016Cryo-electron tomography (cryo-ET) has emerged as a leading technique for three-dimensional visualization of large macromolecular complexes and their conformational... (Review)
Review
Cryo-electron tomography (cryo-ET) has emerged as a leading technique for three-dimensional visualization of large macromolecular complexes and their conformational changes in their native cellular environment. However, the resolution and potential applications of cryo-ET are fundamentally limited by specimen thickness, preventing high-resolution in situ visualization of macromolecular structures in many bacteria (such as Escherichia coli and Salmonella enterica). Minicells, which were discovered nearly 50 years ago, have recently been exploited as model systems to visualize molecular machines in situ, due to their smaller size and other unique properties. In this review, we discuss strategies for producing minicells and highlight their use in the study of chemotactic signaling, protein secretion, and DNA translocation. In combination with powerful genetic tools and advanced imaging techniques, minicells provide a springboard for in-depth structural studies of bacterial macromolecular complexes in situ and therefore offer a unique approach for gaining novel structural insights into many important processes in microbiology.
Topics: Cells; Clinical Laboratory Techniques; Cryoelectron Microscopy; Macromolecular Substances
PubMed: 26833418
DOI: 10.1128/JB.00901-15