Hands on Methods for High Resolution Cryo-Electron Microscopy Structures of Heterogeneous Macromolecular Complexes

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.

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Cryo-EM Is a Powerful Tool, But Helical Applications Can Have Pitfalls

Soft Matter ◽

10.1039/d1sm00282a ◽

2021 ◽

Author(s):

Edward Egelman ◽

Fengbin Wang

Keyword(s):

Electron Microscopy ◽

Structural Biology ◽

Macromolecular Complexes ◽

Atomic Structures ◽

Cryo Electron Microscopy ◽

Main Technique

In structural biology, cryo-electron microscopy (cryo-EM) has emerged as the main technique for determining the atomic structures of macromolecular complexes. This has largely been due to the introduction of direct...

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Practical Experience with Hole-Free Phase Plates for Cryo Electron Microscopy

Microscopy and Microanalysis ◽

10.1017/s143192761601196x ◽

2016 ◽

Vol 22 (6) ◽

pp. 1316-1328 ◽

Cited By ~ 7

Author(s):

Michael Marko ◽

Chyongere Hsieh ◽

Eric Leith ◽

David Mastronarde ◽

Sohei Motoki

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Practical Experience ◽

Phase Plate ◽

Native State ◽

Automated Data Collection ◽

Cryo Electron Microscopy ◽

Transmission Electron ◽

Phase Plates ◽

Set Up

AbstractPhase plate (PP) imaging has proven to be valuable in transmission cryo electron microscopy of unstained, native-state biological specimens. Many PP types have been described, however until the recent implementation of the “hole-free” phase plate (HFPP), imaging has been challenging. We found the HFPP to be simple to construct and to set up in the transmission electron microscopy, but care in implementing automated data collection is needed. Performance may be variable, both initially and over time, thus it is important to monitor and evaluate image quality by observing the power spectrum. We found that while some HFPPs gave transfer to high resolution without CTF oscillation, most reached high resolution when operated with modest defocus.

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A 2.8-Angstrom-Resolution Cryo-Electron Microscopy Structure of Human Parechovirus 3 in Complex with Fab from a Neutralizing Antibody

Journal of Virology ◽

10.1128/jvi.01597-18 ◽

2018 ◽

Vol 93 (4) ◽

Cited By ~ 5

Author(s):

Aušra Domanska ◽

Justin W. Flatt ◽

Joonas J. J. Jukonen ◽

James A. Geraets ◽

Sarah J. Butcher

Keyword(s):

Electron Microscopy ◽

Monoclonal Antibody ◽

High Resolution ◽

Cultured Cells ◽

Human Monoclonal Antibody ◽

Neutralizing Antibody ◽

Molecular Diagnostic ◽

Diagnostic Tools ◽

Human Parechovirus ◽

Cryo Electron Microscopy

ABSTRACTHuman parechovirus 3 (HPeV3) infection is associated with sepsis characterized by significant immune activation and subsequent tissue damage in neonates. Strategies to limit infection have been unsuccessful due to inadequate molecular diagnostic tools for early detection and the lack of a vaccine or specific antiviral therapy. Toward the latter, we present a 2.8-Å-resolution structure of HPeV3 in complex with fragments from a neutralizing human monoclonal antibody, AT12-015, using cryo-electron microscopy (cryo-EM) and image reconstruction. Modeling revealed that the epitope extends across neighboring asymmetric units with contributions from capsid proteins VP0, VP1, and VP3. Antibody decoration was found to block binding of HPeV3 to cultured cells. Additionally, at high resolution, it was possible to model a stretch of RNA inside the virion and, from this, identify the key features that drive and stabilize protein-RNA association during assembly.IMPORTANCEHuman parechovirus 3 (HPeV3) is receiving increasing attention as a prevalent cause of sepsis-like symptoms in neonates, for which, despite the severity of disease, there are no effective treatments available. Structural and molecular insights into virus neutralization are urgently needed, especially as clinical cases are on the rise. Toward this goal, we present the first structure of HPeV3 in complex with fragments from a neutralizing monoclonal antibody. At high resolution, it was possible to precisely define the epitope that, when targeted, prevents virions from binding to cells. Such an atomic-level description is useful for understanding host-pathogen interactions and viral pathogenesis mechanisms and for finding potential cures for infection and disease.

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Computational methods for analyzing conformational variability of macromolecular complexes from cryo-electron microscopy images

Current Opinion in Structural Biology ◽

10.1016/j.sbi.2016.12.011 ◽

2017 ◽

Vol 43 ◽

pp. 114-121 ◽

Cited By ~ 17

Author(s):

Slavica Jonić

Keyword(s):

Electron Microscopy ◽

Computational Methods ◽

Macromolecular Complexes ◽

Conformational Variability ◽

Cryo Electron Microscopy ◽

Microscopy Images

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The structure of the yeast Ctf3 complex

eLife ◽

10.7554/elife.48215 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 5

Author(s):

Stephen M Hinshaw ◽

Andrew N Dates ◽

Stephen C Harrison

Keyword(s):

Electron Microscopy ◽

Cell Cycle ◽

High Resolution ◽

Atomic Model ◽

Kinetochore Assembly ◽

Cryo Electron Microscopy ◽

Molecular Interpretation ◽

Spindle Microtubules ◽

And Function ◽

Assembly Site

Kinetochores are the chromosomal attachment points for spindle microtubules. They are also signaling hubs that control major cell cycle transitions and coordinate chromosome folding. Most well-studied eukaryotes rely on a conserved set of factors, which are divided among two loosely-defined groups, for these functions. Outer kinetochore proteins contact microtubules or regulate this contact directly. Inner kinetochore proteins designate the kinetochore assembly site by recognizing a specialized nucleosome containing the H3 variant Cse4/CENP-A. We previously determined the structure, resolved by cryo-electron microscopy (cryo-EM), of the yeast Ctf19 complex (Ctf19c, homologous to the vertebrate CCAN), providing a high-resolution view of inner kinetochore architecture (Hinshaw and Harrison, 2019). We now extend these observations by reporting a near-atomic model of the Ctf3 complex, the outermost Ctf19c sub-assembly seen in our original cryo-EM density. The model is sufficiently well-determined by the new data to enable molecular interpretation of Ctf3 recruitment and function.

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Rapid high-resolution structure analysis of small, biotechnologically relevant enzymes by cryo-electron microscopy

10.1101/2021.06.15.448552 ◽

2021 ◽

Author(s):

Nicole Dimos ◽

Carl P.O. Helmer ◽

Andrea M. Chanique ◽

Markus C. Wahl ◽

Robert Kourist ◽

...

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Structural Biology ◽

Structure Analysis ◽

Cryo Electron Microscopy ◽

High Resolution Structure ◽

Fast Development ◽

Pharmaceutical Industries ◽

Exquisite Specificity ◽

Resolution Structure

Enzyme catalysis has emerged as a key technology for developing efficient, sustainable processes in the chemical, biotechnological and pharmaceutical industries. Plants provide large and diverse pools of biosynthetic enzymes that facilitate complex reactions, such as the formation of intricate terpene carbon skeletons, with exquisite specificity. High-resolution structural analysis of these enzymes is crucial to understand their mechanisms and modulate their properties by targeted engineering. Although cryo-electron microscopy (cryo-EM) has revolutionized structural biology, its applicability to high-resolution structure analysis of comparatively small enzymes is so far largely unexplored. Here, we show that cryo-EM can reveal the structures of ~120 kDa plant borneol dehydrogenases at or below 2 Å resolution, paving the way for the fast development of new biocatalysts that provide access to bioactive terpenes and terpenoids.

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