High-resolution cryo-EM structure of urease from the pathogen Yersinia enterocolitica

AbstractUrease converts urea into ammonia and carbon dioxide and makes urea available as a nitrogen source for all forms of life except animals. In human bacterial pathogens, ureases also aid in the invasion of acidic environments such as the stomach by raising the surrounding pH. Here, we report the structure of urease from the pathogen Yersinia enterocolitica at better than 2 Å resolution from cryo-electron microscopy. Y. enterocolitica urease is a dodecameric assembly of a trimer of three protein chains, ureA, ureB and ureC. The high data quality enables detailed visualization of the urease bimetal active site and of the impact of radiation damage. Our data are of sufficient quality to support drug development efforts.

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High-resolution cryo-EM structure of urease from the pathogen Yersinia enterocolitica

Nature Communications ◽

10.1038/s41467-020-18870-2 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Ricardo D. Righetto ◽

Leonie Anton ◽

Ricardo Adaixo ◽

Roman P. Jakob ◽

Jasenko Zivanov ◽

...

Keyword(s):

Electron Microscopy ◽

Carbon Dioxide ◽

High Resolution ◽

Drug Development ◽

Yersinia Enterocolitica ◽

Active Site ◽

High Data ◽

Cryo Electron Microscopy ◽

Acidic Environments ◽

The Impact

AbstractUrease converts urea into ammonia and carbon dioxide and makes urea available as a nitrogen source for all forms of life except animals. In human bacterial pathogens, ureases also aid in the invasion of acidic environments such as the stomach by raising the surrounding pH. Here, we report the structure of urease from the pathogen Yersinia enterocolitica at 2 Å resolution from cryo-electron microscopy. Y. enterocolitica urease is a dodecameric assembly of a trimer of three protein chains, ureA, ureB and ureC. The high data quality enables detailed visualization of the urease bimetal active site and of the impact of radiation damage. The obtained structure is of sufficient quality to support drug development efforts.

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Cryo-electron microscopy structures of pyrene-labeled ADP-Pi- and ADP-actin filaments

10.1101/2020.08.26.268748 ◽

2020 ◽

Author(s):

Steven Z. Chou ◽

Thomas D. Pollard

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Active Site ◽

Actin Filaments ◽

Hydrophobic Cavity ◽

Cryo Electron Microscopy

AbstractWe report high resolution cryo-electron microscopy structures of actin filaments with N-1-pyrene conjugated to cysteine 374 and either ADP (3.2 Å) or ADP-phosphate (3.0 Å) in the active site. Polymerization buries pyrene in a hydrophobic cavity between subunits along the long-pitch helix with only minor differences in conformation compared with native actin filaments. These structures explain how polymerization increases the fluorescence 20-fold, how myosin and cofilin binding to filaments reduces the fluorescence and how profilin binding to actin monomers increases the fluorescence.

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Redox-coupled proton pumping drives carbon concentration in the photosynthetic complex I

Nature Communications ◽

10.1038/s41467-020-14347-4 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 14

Author(s):

Jan M. Schuller ◽

Patricia Saura ◽

Jacqueline Thiemann ◽

Sandra K. Schuller ◽

Ana P. Gamiz-Hernandez ◽

...

Keyword(s):

Electron Microscopy ◽

Carbon Dioxide ◽

Carbon Concentration ◽

Active Site ◽

Complex I ◽

Organic Molecules ◽

Reducing Power ◽

Proton Pumping ◽

Photosynthetic Organisms ◽

Cryo Electron Microscopy

AbstractPhotosynthetic organisms capture light energy to drive their energy metabolism, and employ the chemical reducing power to convert carbon dioxide (CO2) into organic molecules. Photorespiration, however, significantly reduces the photosynthetic yields. To survive under low CO2 concentrations, cyanobacteria evolved unique carbon-concentration mechanisms that enhance the efficiency of photosynthetic CO2 fixation, for which the molecular principles have remained unknown. We show here how modular adaptations enabled the cyanobacterial photosynthetic complex I to concentrate CO2 using a redox-driven proton-pumping machinery. Our cryo-electron microscopy structure at 3.2 Å resolution shows a catalytic carbonic anhydrase module that harbours a Zn2+ active site, with connectivity to proton-pumping subunits that are activated by electron transfer from photosystem I. Our findings illustrate molecular principles in the photosynthetic complex I machinery that enabled cyanobacteria to survive in drastically changing CO2 conditions.

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The impact of recent improvements in cryo-electron microscopy technology on the understanding of bacterial ribosome assembly

Nucleic Acids Research ◽

10.1093/nar/gkw1231 ◽

2016 ◽

Vol 45 (3) ◽

pp. 1027-1040 ◽

Cited By ~ 12

Author(s):

Aida Razi ◽

Robert A. Britton ◽

Joaquin Ortega

Keyword(s):

Electron Microscopy ◽

Ribosome Assembly ◽

Cryo Electron Microscopy ◽

Bacterial Ribosome ◽

The Impact

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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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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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