scholarly journals Structure of mycobacterial cytochrome bcc in complex with Q203 and TB47, two anti-TB drug candidates

2021 ◽  
Author(s):  
Shan Zhou ◽  
Weiwei Wang ◽  
Xiaoting Zhou ◽  
Yuying Zhang ◽  
Yuezheng Lai ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Structural Information ◽  
Mycobacterial Infections ◽  
Drug Candidates ◽  
Cryo Electron Microscopy ◽  
Antibiotic Drug ◽  
Binding Pockets ◽  
Quinol Binding ◽  
Clinical Drug

Pathogenic mycobacteria pose a sustained threat to global human health. Recently, cytochrome bcc complexes have gained interest as targets for antibiotic drug development. However, there is currently no structural information for the cytochrome bcc complex from these pathogenic mycobacteria. Here, we report the structures of M. tuberculosis cytochrome bcc alone (2.68 Å resolution) and in complex with clinical drug candidates Q203 (2.67 Å resolution) and TB47 (2.93 Å resolution) determined by single-particle cryo-electron microscopy. M. tuberculosis cytochrome bcc forms a dimeric assembly with endogenous menaquinone/menaquinol bound at the quinone/quinol binding pockets. Q203 and TB47 are bound to the quinol-binding site. Hydrogen bonds are formed between the inhibitor and the side chains of QcrBThr313 and QcrBGlu314, residues that are conserved across pathogenic mycobacteria. These high-resolution structures provide a basis for the design of new mycobacterial cytochrome bcc inhibitors that could be developed into broad spectrum drugs to treat mycobacterial infections.

scholarly journals Structure of Mycobacterium tuberculosis cytochrome bcc in complex with Q203 and TB47, two anti-TB drug candidates

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Shan Zhou ◽  
Weiwei Wang ◽  
Xiaoting Zhou ◽  
Yuying Zhang ◽  
Yuezheng Lai ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Structural Information ◽  
Mycobacterial Infections ◽  
Drug Candidates ◽  
Cryo Electron Microscopy ◽  
Antibiotic Drug ◽  
Binding Pockets ◽  
High Resolution Images ◽  
Quinol Binding ◽  
Clinical Drug

Pathogenic mycobacteria pose a sustained threat to global human health. Recently, cytochrome bcc complexes have gained interest as targets for antibiotic drug development. However, there is currently no structural information for the cytochrome bcc complex from these pathogenic mycobacteria. Here, we report the structures of Mycobacterium tuberculosis cytochrome bcc alone (2.68 Å resolution) and in complex with clinical drug candidates Q203 (2.67 Å resolution) and TB47 (2.93 Å resolution) determined by single-particle cryo-electron microscopy. M. tuberculosis cytochrome bcc forms a dimeric assembly with endogenous menaquinone/menaquinol bound at the quinone/quinol-binding pockets. We observe Q203 and TB47 bound at the quinol-binding site and stabilized by hydrogen bonds with the side chains of QcrBThr313 and QcrBGlu314, residues that are conserved across pathogenic mycobacteria. These high-resolution images provide a basis for the design of new mycobacterial cytochrome bcc inhibitors that could be developed into broad-spectrum drugs to treat mycobacterial infections.

scholarly journals Cryo-EM structure of the bacterial Ton motor subcomplex ExbB–ExbD provides information on structure and stoichiometry

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Herve Celia ◽  
Istvan Botos ◽  
Xiaodan Ni ◽  
Tara Fox ◽  
Natalia De Val ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Electron Microscopy ◽  
High Resolution ◽  
Outer Membrane ◽  
Motor Function ◽  
Structural Information ◽  
Molecular Motor ◽  
Single Copy ◽  
Proton Motive Force ◽  
Cryo Electron Microscopy

Abstract The TonB–ExbB–ExbD molecular motor harnesses the proton motive force across the bacterial inner membrane to couple energy to transporters at the outer membrane, facilitating uptake of essential nutrients such as iron and cobalamine. TonB physically interacts with the nutrient-loaded transporter to exert a force that opens an import pathway across the outer membrane. Until recently, no high-resolution structural information was available for this unique molecular motor. We published the first crystal structure of ExbB–ExbD in 2016 and showed that five copies of ExbB are arranged as a pentamer around a single copy of ExbD. However, our spectroscopic experiments clearly indicated that two copies of ExbD are present in the complex. To resolve this ambiguity, we used single-particle cryo-electron microscopy to show that the ExbB pentamer encloses a dimer of ExbD in its transmembrane pore, and not a monomer as previously reported. The revised stoichiometry has implications for motor function.

scholarly journals Cryo-EM structure of a 40 kDa SAM-IV riboswitch RNA at 3.7 Å resolution

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Kaiming Zhang ◽  
Shanshan Li ◽  
Kalli Kappel ◽  
Grigore Pintilie ◽  
Zhaoming Su ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Ligand Binding ◽  
Small Rnas ◽  
Sulfur Metabolism ◽  
Cryo Electron Microscopy ◽  
Binding Core ◽  
New Antibiotics ◽  
Binding Pockets ◽  
High Flexibility

AbstractSpecimens below 50 kDa have generally been considered too small to be analyzed by single-particle cryo-electron microscopy (cryo-EM). The high flexibility of pure RNAs makes it difficult to obtain high-resolution structures by cryo-EM. In bacteria, riboswitches regulate sulfur metabolism through binding to the S-adenosylmethionine (SAM) ligand and offer compelling targets for new antibiotics. SAM-I, SAM-I/IV, and SAM-IV are the three most commonly found SAM riboswitches, but the structure of SAM-IV is still unknown. Here, we report the structures of apo and SAM-bound SAM-IV riboswitches (119-nt, ~40 kDa) to 3.7 Å and 4.1 Å resolution, respectively, using cryo-EM. The structures illustrate homologies in the ligand-binding core but distinct peripheral tertiary contacts in SAM-IV compared to SAM-I and SAM-I/IV. Our results demonstrate the feasibility of resolving small RNAs with enough detail to enable detection of their ligand-binding pockets and suggest that cryo-EM could play a role in structure-assisted drug design for RNA.

scholarly journals Approaches to altering particle distributions in cryo-electron microscopy sample preparation

2018 ◽  
Vol 74 (6) ◽  
pp. 560-571 ◽  
Author(s):  
Ieva Drulyte ◽  
Rachel M. Johnson ◽  
Emma L. Hesketh ◽  
Daniel L. Hurdiss ◽  
Charlotte A. Scarff ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Sample Preparation ◽  
Single Particle ◽  
Structural Information ◽  
Particle Analysis ◽  
Diverse Range ◽  
Sample Distribution ◽  
High Resolution Data ◽  
Cryo Electron Microscopy

Cryo-electron microscopy (cryo-EM) can now be used to determine high-resolution structural information on a diverse range of biological specimens. Recent advances have been driven primarily by developments in microscopes and detectors, and through advances in image-processing software. However, for many single-particle cryo-EM projects, major bottlenecks currently remain at the sample-preparation stage; obtaining cryo-EM grids of sufficient quality for high-resolution single-particle analysis can require the careful optimization of many variables. Common hurdles to overcome include problems associated with the sample itself (buffer components, labile complexes), sample distribution (obtaining the correct concentration, affinity for the support film), preferred orientation, and poor reproducibility of the grid-making process within and between batches. This review outlines a number of methodologies used within the electron-microscopy community to address these challenges, providing a range of approaches which may aid in obtaining optimal grids for high-resolution data collection.

Progress in high-resolution CRYO-SEM imaging of viral particles

1996 ◽  
Vol 54 ◽  
pp. 818-819
Author(s):  
Ya Chen ◽  
Geoffrey Letchworth ◽  
John White
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
High Resolution ◽  
Structural Information ◽  
Signal To Noise Ratio ◽  
Flexible Structures ◽  
Simian Virus ◽  
Topographic Features ◽  
Viral Particles ◽  
Scanning Electron

Low-temperature high-resolution scanning electron microscopy (cryo-HRSEM) has been successfully utilized to image biological macromolecular complexes at nanometer resolution. Recently, imaging of individual viral particles such as reovirus using cryo-HRSEM or simian virus (SIV) using HRSEM, HV-STEM and AFM have been reported. Although conventional electron microscopy (e.g., negative staining, replica, embedding and section), or cryo-TEM technique are widely used in studying of the architectures of viral particles, scanning electron microscopy presents two major advantages. First, secondary electron signal of SEM represents mostly surface topographic features. The topographic details of a biological assembly can be viewed directly and will not be obscured by signals from the opposite surface or from internal structures. Second, SEM may produce high contrast and signal-to-noise ratio images. As a result of this important feature, it is capable of visualizing not only individual virus particles, but also asymmetric or flexible structures. The 2-3 nm resolution obtained using high resolution cryo-SEM made it possible to provide useful surface structural information of macromolecule complexes within cells and tissues. In this study, cryo-HRSEM is utilized to visualize the distribution of glycoproteins of a herpesvirus.

Atomic chemistry by HREM and image simulations?

1986 ◽  
Vol 44 ◽  
pp. 828-829
Author(s):  
J.M. Howe ◽  
R. Gronsky
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Structural Information ◽  
High Resolution Electron Microscopy ◽  
Resolution Electron ◽  
Image Simulations

The technique of high-resolution electron microscopy (HREM) is invaluable to the materials scientist because it allows examination of microstructural features at levels of resolution that are unobtainable by most other methods. Although the structural information which can be determined by HREM and accompanying image simulations has been well documented in the literature, there have only been a few cases where this technique has been used to reveal the chemistry of individual columns or planes of atoms, as occur in segregated and ordered materials.

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

2016 ◽  
Vol 22 (6) ◽  
pp. 1316-1328 ◽  
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.

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

2018 ◽  
Vol 93 (4) ◽  
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.

scholarly journals The structure of the yeast Ctf3 complex

eLife ◽  
2019 ◽  
Vol 8 ◽  
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.

scholarly journals A resolution record for cryoEM

2021 ◽  
Vol 10 ◽  
Author(s):  
Jonathan Ashmore ◽  
Bridget Carragher ◽  
Peter B Rosenthal ◽  
William Weis
Keyword(s):  
Electron Microscopy ◽  
Image Analysis ◽  
Structure Determination ◽  
Test Specimen ◽  
Structural Information ◽  
Atomic Resolution ◽  
Fast Growing ◽  
Cryo Electron Microscopy ◽  
New Instrument ◽  
Resolution Structure

Cryo electron microscopy (cryoEM) is a fast-growing technique for structure determination. Two recent papers report the first atomic resolution structure of a protein obtained by averaging images of frozen-hydrated biomolecules. They both describe maps of symmetric apoferritin assemblies, a common test specimen, in unprecedented detail. New instrument improvements, different in the two studies, have contributed better images, and image analysis can extract structural information sufficient to resolve individual atomic positions. While true atomic resolution maps will not be routine for most proteins, the studies suggest structures determined by cryoEM will continue to improve, increasing their impact on biology and medicine.

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