Programming new geometry restraints: parallelity of atomic groups

Improvements in structural biology methods, in particular crystallography and cryo-electron microscopy, have created an increased demand for the refinement of atomic models against low-resolution experimental data. One way to compensate for the lack of high-resolution experimental data is to usea prioriinformation about model geometry that can be utilized in refinement in the form of stereochemical restraints or constraints. Here, the definition and calculation of the restraints that can be imposed on planar atomic groups, in particular the angle between such groups, are described. Detailed derivations of the restraint targets and their gradients are provided so that they can be readily implemented in other contexts. Practical implementations of the restraints, and of associated data structures, in theComputational Crystallography Toolbox(cctbx) are presented.

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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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Principal component analysis is limited to low-resolution analysis in cryoEM

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798321002291 ◽

2021 ◽

Vol 77 (6) ◽

Author(s):

Carlos Oscar S. Sorzano ◽

Jose Maria Carazo

Keyword(s):

Electron Microscopy ◽

Principal Component Analysis ◽

High Resolution ◽

Principal Component ◽

Component Analysis ◽

Low Resolution ◽

Cryo Electron Microscopy ◽

Resolution Analysis

Principal component analysis (PCA) has been widely proposed to analyze flexibility and heterogeneity in cryo-electron microscopy (cryoEM). In this paper, it is argued that (i) PCA is an excellent technique to describe continuous flexibility at low resolution (but not so much at high resolution) and (ii) PCA components should be analyzed in a concerted manner (and not independently).

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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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Structural biology techniques: X-ray crystallography, cryo-electron microscopy, and small-angle X-ray scattering

Practical Approaches to Biological Inorganic Chemistry ◽

10.1016/b978-0-444-64225-7.00010-9 ◽

2020 ◽

pp. 375-416

Author(s):

José A. Brito ◽

Margarida Archer

Keyword(s):

Electron Microscopy ◽

Structural Biology ◽

Small Angle ◽

X Ray ◽

X Ray Crystallography ◽

X Ray Scattering ◽

Cryo Electron Microscopy ◽

Ray Scattering

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Single-particle cryo-EM—How did it get here and where will it go

Science ◽

10.1126/science.aat4346 ◽

2018 ◽

Vol 361 (6405) ◽

pp. 876-880 ◽

Cited By ~ 105

Author(s):

Yifan Cheng

Keyword(s):

Electron Microscopy ◽

Structural Biology ◽

Single Particle ◽

Electron Crystallography ◽

The Past ◽

Cryo Electron Microscopy ◽

Technological Advances ◽

The Future ◽

Electron Cryotomography

Cryo–electron microscopy, or simply cryo-EM, refers mainly to three very different yet closely related techniques: electron crystallography, single-particle cryo-EM, and electron cryotomography. In the past few years, single-particle cryo-EM in particular has triggered a revolution in structural biology and has become a newly dominant discipline. This Review examines the fascinating story of its start and evolution over the past 40-plus years, delves into how and why the recent technological advances have been so groundbreaking, and briefly considers where the technique may be headed in the future.

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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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