scholarly journals Structure of the Ebola virus nucleoprotein – RNA complex

2019 ◽  
Author(s):  
Robert N. Kirchdoerfer ◽  
Erica Ollmann Saphire ◽  
Andrew B. Ward
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Ebola Virus ◽  
Viral Proteins ◽  
Cryo Electron Microscopy ◽  
Emerging Virus ◽  
Deadly Disease ◽  
Rna Complex ◽  
Viral Nucleocapsid

AbstractEbola virus is an emerging virus capable of causing a deadly disease in humans. Replication, transcription and packaging of the viral genome is carried out by the viral nucleocapsid. The nucleocapsid is a complex of the viral nucleoprotein, RNA and several other viral proteins. The nucleoprotein NP forms large, RNA-bound, helical filaments and acts as a scaffold for additional viral proteins. The 3.1 Å single-particle cryo-electron microscopy structure of the nucleoprotein-RNA helical filament presented here resembles previous structures determined at lower resolution while providing improved molecular details of protein-protein and protein-RNA interactions. The higher resolution of the structure presented here will facilitate the design and characterization of novel and specific Ebola virus therapeutics targeting the nucleocapsid.SynopsisThe 3.1 Å single-particle cryo-electron microscopy structure of the RNA-bound, Ebola virus nucleoprotein helical filament provides molecular details of protein-protein and protein-RNA interactions.

scholarly journals Cryo-EM structure of the Ebola virus nucleoprotein–RNA complex

2019 ◽  
Vol 75 (5) ◽  
pp. 340-347 ◽  
Author(s):  
Robert N. Kirchdoerfer ◽  
Erica Ollmann Saphire ◽  
Andrew B. Ward
Keyword(s):  
Viral Genome ◽  
Ebola Virus ◽  
Viral Proteins ◽  
Cryo Electron Microscopy ◽  
Emerging Virus ◽  
Deadly Disease ◽  
Rna Complex ◽  
Resolution Single ◽  
Viral Nucleocapsid

Ebola virus is an emerging virus that is capable of causing a deadly disease in humans. Replication, transcription and packaging of the viral genome are carried out by the viral nucleocapsid. The nucleocapsid is a complex of the viral nucleoprotein, RNA and several other viral proteins. The nucleoprotein forms large, RNA-bound, helical filaments and acts as a scaffold for additional viral proteins. The 3.1 Å resolution single-particle cryo-electron microscopy structure of the nucleoprotein–RNA helical filament presented here resembles previous structures determined at lower resolution, while providing improved molecular details of protein–protein and protein–RNA interactions. The higher resolution of the structure presented here will facilitate the design and characterization of novel and specific Ebola virus therapeutics targeting the nucleocapsid.

Uncovering the structures of modular polyketide synthases

2015 ◽  
Vol 32 (3) ◽  
pp. 436-453 ◽  
Author(s):  
Kira J. Weissman
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Structural Characterization ◽  
Small Angle ◽  
Single Particle ◽  
Polyketide Synthases ◽  
X Ray ◽  
X Ray Scattering ◽  
Cryo Electron Microscopy

This review covers a breakthrough in the structural biology of the gigantic modular polyketide synthases (PKS): the structural characterization of intact modules by single-particle cryo-electron microscopy and small-angle X-ray scattering.

scholarly journals Structure of the Ebola Virus Nucleocapsid Core by Single Particle Cryo-Electron Microscopy

2016 ◽  
Vol 22 (S3) ◽  
pp. 66-67 ◽  
Author(s):  
Yukihiko Sugita ◽  
Yoshihiro Kawaoka ◽  
Takeshi Noda ◽  
Matthias Wolf
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Ebola Virus ◽  
Cryo Electron Microscopy

scholarly journals Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids

2016 ◽  
Author(s):  
Joost Snijder ◽  
Andrew J. Borst ◽  
Annie Dosey ◽  
Alexandra C. Walls ◽  
Anika Burrell ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Particle Density ◽  
Protein Complexes ◽  
Cost Effective ◽  
Carbon Support ◽  
Cryo Electron Microscopy ◽  
Sample Application ◽  
Single Field

Single particle cryo-electron microscopy (cryoEM) is becoming widely adopted as a tool for structural characterization of biomolecules at near-atomic resolution. Vitrification of the sample to obtain a dense distribution of particles within a single field of view remains a major bottleneck for the success of such experiments. Here, we describe a simple and cost-effective method to increase the density of frozen-hydrated particles on grids with holey carbon support films. It relies on performing multiple rounds of sample application and blotting prior to plunge freezing in liquid ethane. We show that this approach is generally applicable and significantly increases particle density for a range of samples, such as small protein complexes, viruses and filamentous assemblies. The method is versatile, easy to implement, minimizes sample requirements and can enable characterization of samples that would otherwise resist structural studies using single particle cryoEM.

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

Science ◽  
2018 ◽  
Vol 361 (6405) ◽  
pp. 876-880 ◽  
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.

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 LRRC8A homohexameric channels poorly recapitulate VRAC regulation and pharmacology

2020 ◽  
Author(s):  
Toshiki Yamada ◽  
Eric E. Figueroa ◽  
Jerod S. Denton ◽  
Kevin Strange
Keyword(s):  
Electron Microscopy ◽  
Voltage Dependence ◽  
Function Analysis ◽  
Cell Swelling ◽  
Independent Manner ◽  
Cryo Electron Microscopy ◽  
Homologous Position ◽  
Pore Properties ◽  
Hct116 Cells

Swelling-activated VRACs are heterohexameric channels comprising LRRC8A and at least one other LRRC8 paralog. Cryo-electron microscopy (EM) structures of non-native LRRC8A and LRRC8D homohexamers have been described. We demonstrate here that LRRC8A homohexamers poorly recapitulate VRAC functional properties. Unlike VRACs, LRRC8A channels heterologously expressed in Lrr8c-/- HCT116 cells are poorly activated by low intracellular ionic strength (µ) and insensitive to cell swelling with normal µ. Combining low µ with swelling modestly activates LRRC8A allowing characterization of pore properties. VRACs are strongly inhibited by 10 mM DCPIB in a voltage-independent manner. In contrast, DCPIB block of LRRC8A is weak and voltage sensitive. Cryo-EM structures indicate that DCPIB block is dependent on arginine 103. Consistent with this, LRRC8A R103F mutants are insensitive to DCPIB. However, a LRRC8 chimeric channel in which R103 is replaced by a leucine at the homologous position is inhibited ~90% by 10 mM DCPIB in a voltage-independent manner. Coexpression of LRRC8A and LRRC8C gives rise to channels with DCPIB sensitivity that is strongly µ-dependent. At normal intracellular µ, LRRC8A+LRRC8C heterohexamers exhibit strong, voltage-independent DCPIB block that is insensitive to R103F. DCPIB inhibition is greatly reduced and exhibits voltage dependence with low intracellular µ. The R103F mutation has no effect on maximal DCPIB inhibition but eliminates voltage-dependence under low µ conditions. Our findings demonstrate that the LRRC8A cryo-EM structure and the use of heterologously expressed LRRC8 heterohexameric channels pose significant limitations for VRAC mutagenesis-based structure-function analysis. Native VRAC function is most closely mimicked by chimeric LRRC8 homohexameric channels.

scholarly journals A general mechanism of ribosome dimerization revealed by single-particle cryo-electron microscopy

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Linda E. Franken ◽  
Gert T. Oostergetel ◽  
Tjaard Pijning ◽  
Pranav Puri ◽  
Valentina Arkhipova ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
General Mechanism ◽  
Cryo Electron Microscopy
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