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.

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.

Domain topology of human Rasal

2017 ◽  
Vol 399 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Jorge Cuellar ◽  
José María Valpuesta ◽  
Alfred Wittinghofer ◽  
Begoña Sot
Keyword(s):  
Electron Microscopy ◽  
Small Gtpase ◽  
Full Length ◽  
Gtpase Activating Protein ◽  
Ras Gtpase ◽  
Cryo Electron Microscopy ◽  
Domain Protein ◽  
Negative Staining Electron Microscopy ◽  
Resolution Structure

AbstractRasal is a modular multi-domain protein of the GTPase-activating protein 1 (GAP1) family; its four known members, GAP1m, Rasal, GAP1IP4BPand Capri, have a Ras GTPase-activating domain (RasGAP). This domain supports the intrinsically slow GTPase activity of Ras by actively participating in the catalytic reaction. In the case of Rasal, GAP1IP4BPand Capri, their remaining domains are responsible for converting the RasGAP domains into dual Ras- and Rap-GAPs, via an incompletely understood mechanism. Although Rap proteins are small GTPase homologues of Ras, their catalytic residues are distinct, which reinforces the importance of determining the structure of full-length GAP1 family proteins. To date, these proteins have not been crystallized, and their size is not adequate for nuclear magnetic resonance (NMR) or for high-resolution cryo-electron microscopy (cryoEM). Here we present the low resolution structure of full-length Rasal, obtained by negative staining electron microscopy, which allows us to propose a model of its domain topology. These results help to understand the role of the different domains in controlling the dual GAP activity of GAP1 family proteins.

scholarly journals The Primary Enveloped Virion of Herpes Simplex Virus 1: Its Role in Nuclear Egress

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
William W. Newcomb ◽  
Juan Fontana ◽  
Dennis C. Winkler ◽  
Naiqian Cheng ◽  
J. Bernard Heymann ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Nuclear Membrane ◽  
Molecular Mechanisms ◽  
Weak Interactions ◽  
Nuclear Pore ◽  
Nuclear Egress ◽  
Cryo Electron Microscopy ◽  
Perinuclear Space ◽  
Infected Cells ◽  
Hsv 1

ABSTRACTMany viruses migrate between different cellular compartments for successive stages of assembly. The HSV-1 capsid assembles in the nucleus and then transfers into the cytoplasm. First, the capsid buds through the inner nuclear membrane, becoming coated with nuclear egress complex (NEC) protein. This yields a primary enveloped virion (PEV) whose envelope fuses with the outer nuclear membrane, releasing the capsid into the cytoplasm. We investigated the associated molecular mechanisms by isolating PEVs from US3-null-infected cells and imaging them by cryo-electron microscopy and tomography. (pUS3 is a viral protein kinase in whose absence PEVs accumulate in the perinuclear space.) Unlike mature extracellular virions, PEVs have very few glycoprotein spikes. PEVs are ~20% smaller than mature virions, and the little space available between the capsid and the NEC layer suggests that most tegument proteins are acquired later in the egress pathway. Previous studies have proposed that NEC is organized as hexamers in honeycomb arrays in PEVs, but we find arrays of heptameric rings in extracts from US3-null-infected cells. In a PEV, NEC contacts the capsid predominantly via the pUL17/pUL25 complexes which are located close to the capsid vertices. Finally, the NEC layer dissociates from the capsid as it leaves the nucleus, possibly in response to pUS3-mediated phosphorylation. Overall, nuclear egress emerges as a process driven by a program of multiple weak interactions.IMPORTANCEOn its maturation pathway, the newly formed HSV-1 nucleocapsid must traverse the nuclear envelope, while respecting the integrity of that barrier. Nucleocapsids (125 nm in diameter) are too large to pass through the nuclear pore complexes that conduct most nucleocytoplasmic traffic. It is now widely accepted that the process involves envelopment/de-envelopment of a key intermediate—the primary enveloped virion. In wild-type infections, PEVs are short-lived, which has impeded study. Using a mutant that accumulates PEVs in the perinuclear space, we were able to isolate PEVs in sufficient quantity for structural analysis by cryo-electron microscopy and tomography. The findings not only elucidate the maturation pathway of an important human pathogen but also have implications for cellular processes that involve the trafficking of large macromolecular complexes.

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.

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 Molecular mechanisms of gating in the calcium-activated chloride channel bestrophin

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Alexandria N Miller ◽  
George Vaisey ◽  
Stephen B Long
Keyword(s):  
Electron Microscopy ◽  
Ion Channels ◽  
Molecular Mechanisms ◽  
Peptide Binding ◽  
Retinal Disease ◽  
Structural Similarity ◽  
Cryo Electron Microscopy ◽  
Cl Channels ◽  
Pore Lining ◽  
Protein Shell

Bestrophin (BEST1-4) ligand-gated chloride (Cl-) channels are activated by calcium (Ca2+). Mutation of BEST1 causes retinal disease. Partly because bestrophin channels have no sequence or structural similarity to other ion channels, the molecular mechanisms underlying gating are unknown. Here, we present a series of cryo-electron microscopy structures of chicken BEST1, determined at 3.1 Å resolution or better, that represent the channel’s principal gating states. Unlike other channels, opening of the pore is due to the repositioning of tethered pore-lining helices within a surrounding protein shell that dramatically widens a neck of the pore through a concertina of amino acid rearrangements. The neck serves as both the activation and the inactivation gate. Ca2+ binding instigates opening of the neck through allosteric means whereas inactivation peptide binding induces closing. An aperture within the otherwise wide pore controls anion permeability. The studies define a new molecular paradigm for gating among ligand-gated ion channels.

Sign in / Sign up

Export Citation Format

Share Document