C3-P-13Single Particle Analysis ofThermus ThermophilusV-ATPase by Cryo-Electron Microscopy

Microscopy ◽  
2015 ◽  
Vol 64 (suppl 1) ◽  
pp. i132.2-i132
Author(s):  
Kaoru Mitsuoka ◽  
Atsuko Nakanishi ◽  
Nao Takeuchi ◽  
Jun-ichi Kishikawa ◽  
Ken Yokoyama
Keyword(s):  
Electron Microscopy ◽  
Particle Analysis ◽  
Cryo Electron Microscopy

scholarly journals Cryo–electron microscopy structure of the antidiuretic hormone arginine-vasopressin V2 receptor signaling complex

2021 ◽  
Vol 7 (21) ◽  
pp. eabg5628
Author(s):  
Julien Bous ◽  
Hélène Orcel ◽  
Nicolas Floquet ◽  
Cédric Leyrat ◽  
Joséphine Lai-Kee-Him ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Antidiuretic Hormone ◽  
Arginine Vasopressin ◽  
Molecular Mechanisms ◽  
Particle Analysis ◽  
Resonance Spectroscopy ◽  
Gpcr Signaling ◽  
Signaling Complex ◽  
Cryo Electron Microscopy ◽  
V2 Receptor

The antidiuretic hormone arginine-vasopressin (AVP) forms a signaling complex with the V2 receptor (V2R) and the Gs protein, promoting kidney water reabsorption. Molecular mechanisms underlying activation of this critical G protein–coupled receptor (GPCR) signaling system are still unknown. To fill this gap of knowledge, we report here the cryo–electron microscopy structure of the AVP-V2R-Gs complex. Single-particle analysis revealed the presence of three different states. The two best maps were combined with computational and nuclear magnetic resonance spectroscopy constraints to reconstruct two structures of the ternary complex. These structures differ in AVP and Gs binding modes. They reveal an original receptor-Gs interface in which the Gαs subunit penetrates deep into the active V2R. The structures help to explain how V2R R137H or R137L/C variants can lead to two severe genetic diseases. Our study provides important structural insights into the function of this clinically relevant GPCR signaling complex.

scholarly journals In situ structure determination using single particle cryo-electron microscopy images

2020 ◽  
Author(s):  
Jing Cheng ◽  
Bufan Li ◽  
Long Si ◽  
Xinzheng Zhang
Keyword(s):  
Electron Microscopy ◽  
Structure Determination ◽  
Single Particle ◽  
Structure Refinement ◽  
Target Function ◽  
Target Protein ◽  
Particle Analysis ◽  
Cryo Electron Microscopy ◽  
Tilt Series

AbstractCryo-electron microscopy (cryo-EM) tomography is a powerful tool for in situ structure determination. However, this method requires the acquisition of tilt series, and its time consuming throughput of acquiring tilt series severely slows determination of in situ structures. By treating the electron densities of non-target protein as non-Gaussian distributed noise, we developed a new target function that greatly improves the efficiency of the recognition of the target protein in a single cryo-EM image without acquiring tilt series. Moreover, we developed a sorting function that effectively eliminates the false positive detection, which not only improves the resolution during the subsequent structure refinement procedure but also allows using homolog proteins as models to recognize the target protein. Together, we developed an in situ single particle analysis (isSPA) method. Our isSPA method was successfully applied to solve structures of glycoproteins on the surface of a non-icosahedral virus and Rubisco inside the carboxysome. The cryo-EM data from both samples were collected within 24 hours, thus allowing fast and simple structural determination in situ.

scholarly journals A national facility for biological cryo-electron microscopy

2015 ◽  
Vol 71 (1) ◽  
pp. 127-135 ◽  
Author(s):  
Helen R. Saibil ◽  
Kay Grünewald ◽  
David I. Stuart
Keyword(s):  
Electron Microscopy ◽  
Three Dimensional ◽  
Particle Analysis ◽  
Dimensional Electron ◽  
Cryo Electron Microscopy ◽  
Cellular Context ◽  
Molecular Machinery ◽  
User Friendly ◽  
Integrated Structural Biology ◽  
Expert Support

Three-dimensional electron microscopy is an enormously powerful tool for structural biologists. It is now able to provide an understanding of the molecular machinery of cells, disease processes and the actions of pathogenic organisms from atomic detail through to the cellular context. However, cutting-edge research in this field requires very substantial resources for equipment, infrastructure and expertise. Here, a brief overview is provided of the plans for a UK national three-dimensional electron-microscopy facility for integrated structural biology to enable internationally leading research on the machinery of life. State-of-the-art equipment operated with expert support will be provided, optimized for both atomic-level single-particle analysis of purified macromolecules and complexes and for tomography of cell sections. The access to and organization of the facility will be modelled on the highly successful macromolecular crystallography (MX) synchrotron beamlines, and will be embedded at the Diamond Light Source, facilitating the development of user-friendly workflows providing near-real-time experimental feedback.

scholarly journals In situ structure determination using single particle cryo-electron microscopy images

2020 ◽  
Author(s):  
Jing Cheng ◽  
Bufan Li ◽  
Long Si ◽  
Xinzheng Zhang
Keyword(s):  
Electron Microscopy ◽  
Structure Determination ◽  
Single Particle ◽  
Structure Refinement ◽  
Target Function ◽  
Target Protein ◽  
Particle Analysis ◽  
Cryo Electron Microscopy ◽  
Tilt Series

Abstract Cryo-electron microscopy (cryo-EM) tomography is a powerful tool for in situ structure determination. However, this method requires the acquisition of tilt series, and its time consuming throughput of acquiring tilt series severely slows determination of in situ structures. By treating the electron densities of non-target protein as non-Gaussian distributed noise, we developed a new target function that greatly improves the efficiency of the recognition of the target protein in a single cryo-EM image without acquiring tilt series. Moreover, we developed a sorting function that effectively eliminates the false positive detection, which not only improves the resolution during the subsequent structure refinement procedure but also allows using homolog proteins as models to recognize the target protein. Together, we developed an in situ single particle analysis (isSPA) method. Our isSPA method was successfully applied to solve structures of glycoproteins on the surface of a non-icosahedral virus and Rubisco inside the carboxysome. The cryo-EM data from both samples were collected within 24 hours, thus allowing fast and simple structural determination in situ.

scholarly journals Cryo-Electron Microscopy of Arabidopsis thaliana Phytochrome A in Its Pr State Reveals Head-to-Head Homodimeric Architecture

2021 ◽  
Vol 12 ◽  
Author(s):  
Weixiao Yuan Wahlgren ◽  
David Golonka ◽  
Sebastian Westenhoff ◽  
Andreas Möglich
Keyword(s):  
Electron Microscopy ◽  
Arabidopsis Thaliana ◽  
Electron Density ◽  
Molecular Mechanisms ◽  
Signal Propagation ◽  
Full Length ◽  
Particle Analysis ◽  
Phytochrome A ◽  
Cryo Electron Microscopy ◽  
Bacterial Phytochromes

Phytochrome photoreceptors regulate vital adaptations of plant development, growth, and physiology depending on the ratio of red and far-red light. The light-triggered Z/E isomerization of a covalently bound bilin chromophore underlies phytochrome photoconversion between the red-absorbing Pr and far-red-absorbing Pfr states. Compared to bacterial phytochromes, the molecular mechanisms of signal propagation to the C-terminal module and its regulation are little understood in plant phytochromes, not least owing to a dearth of structural information. To address this deficit, we studied the Arabidopsis thaliana phytochrome A (AtphyA) at full length by cryo-electron microscopy (cryo-EM). Following heterologous expression in Escherichia coli, we optimized the solvent conditions to overcome protein aggregation and thus obtained photochemically active, near-homogenous AtphyA. We prepared grids for cryo-EM analysis of AtphyA in its Pr state and conducted single-particle analysis. The resulting two-dimensional class averages and the three-dimensional electron density map at 17 Å showed a homodimeric head-to-head assembly of AtphyA. Docking of domain structures into the electron density revealed a separation of the AtphyA homodimer at the junction of its photosensor and effector modules, as reflected in a large void in the middle of map. The overall architecture of AtphyA resembled that of bacterial phytochromes, thus hinting at commonalities in signal transduction and mechanism between these receptors. Our work paves the way toward future studies of the structure, light response, and interactions of full-length phytochromes by cryo-EM.

Using Cryo-EM to Investigate Bacterial Secretion Systems

2018 ◽  
Vol 72 (1) ◽  
pp. 231-254 ◽  
Author(s):  
Chiara Rapisarda ◽  
Matteo Tassinari ◽  
Francesca Gubellini ◽  
Rémi Fronzes
Keyword(s):  
Electron Microscopy ◽  
Particle Analysis ◽  
Single Particle Analysis ◽  
Secretion Systems ◽  
Extracellular Milieu ◽  
Cryo Electron Microscopy ◽  
Bacterial Fitness ◽  
High Resolution Images ◽  
The Impact ◽  
Bacterial Secretion

Bacterial secretion systems are responsible for releasing macromolecules to the extracellular milieu or directly into other cells. These membrane complexes are associated with pathogenicity and bacterial fitness. Understanding of these large assemblies has exponentially increased in the last few years thanks to electron microscopy. In fact, a revolution in this field has led to breakthroughs in characterizing the structures of secretion systems and other macromolecular machineries so as to obtain high-resolution images of complexes that could not be crystallized. In this review, we give a brief overview of structural advancements in the understanding of secretion systems, focusing in particular on cryo–electron microscopy, whether tomography or single-particle analysis. We describe how such techniques have contributed to knowledge of the mechanism of macromolecule secretion in bacteria and the impact they will have in the future.

scholarly journals Good Vibrations: Structural Remodeling of Maturing Yeast Pre-40S Ribosomal Particles Followed by Cryo-Electron Microscopy

Molecules ◽  
2020 ◽  
Vol 25 (5) ◽  
pp. 1125 ◽  
Author(s):  
Ramtin Shayan ◽  
Dana Rinaldi ◽  
Natacha Larburu ◽  
Laura Plassart ◽  
Stéphanie Balor ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Particle Analysis ◽  
Ribosomal Subunits ◽  
Early Maturation ◽  
Cryo Electron Microscopy ◽  
Molecular Events ◽  
Final Maturation

Assembly of eukaryotic ribosomal subunits is a very complex and sequential process that starts in the nucleolus and finishes in the cytoplasm with the formation of functional ribosomes. Over the past few years, characterization of the many molecular events underlying eukaryotic ribosome biogenesis has been drastically improved by the “resolution revolution” of cryo-electron microscopy (cryo-EM). However, if very early maturation events have been well characterized for both yeast ribosomal subunits, little is known regarding the final maturation steps occurring to the small (40S) ribosomal subunit. To try to bridge this gap, we have used proteomics together with cryo-EM and single particle analysis to characterize yeast pre-40S particles containing the ribosome biogenesis factor Tsr1. Our analyses lead us to refine the timing of the early pre-40S particle maturation steps. Furthermore, we suggest that after an early and structurally stable stage, the beak and platform domains of pre-40S particles enter a “vibrating” or “wriggling” stage, that might be involved in the final maturation of 18S rRNA as well as the fitting of late ribosomal proteins into their mature position.

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