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CryoEM of coliphage PR772 reveals the composition & structure of the elusive vertex complex and the capsid architecture.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
(English)In: eLIFE, E-ISSN 2050-084XArticle in journal (Refereed) Submitted
Abstract [en]

Bacteriophage PR772, a member of the Tectiviridae family, has a 70-nm diameter icosahedral protein capsid that encapsulates a lipid membrane, dsDNA, and various internal proteins. An icosahedrally averaged CryoEM reconstruction of the wild-type virion and a localized reconstruction of the vertex region reveal the composition and the structure of the vertex complex along with new protein conformations that play a vital role in maintaining the capsid architecture of the virion. The overall resolution of the virion is 2.75 Å, while the resolution of the protein capsid is 2.3 Å. The conventional penta-symmetron formed by the capsomeres is replaced by a large vertex complex in the pseudo T=25 capsid. All the vertices contain the host-recognition protein, P5; two of these vertices show the presence of the receptor-binding protein, P2. The 3D structure of the vertex complex shows interactions with the viral membrane, indicating a possible mechanism for viral infection.

Keywords [en]
PR772, Phage, Bacteriophage, PRD1, Vertex Complex, Penton, heteropentamer
National Category
Structural Biology
Identifiers
URN: urn:nbn:se:uu:diva-391669OAI: oai:DiVA.org:uu-391669DiVA, id: diva2:1345494
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationEU, European Research CouncilAvailable from: 2019-08-25 Created: 2019-08-25 Last updated: 2019-08-25
In thesis
1. Structural Studies of Large dsDNA Viruses using Single Particle Methods
Open this publication in new window or tab >>Structural Studies of Large dsDNA Viruses using Single Particle Methods
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Structural studies of large biological assemblies pose a unique problem due to their size, complexity and heterogeneity. Conventional methods like x-ray crystallography, NMR, etc. are limited in their ability to address these issues. To overcome some of these limitations, single particle methods were used. In these methods, each particle image is manipulated individually to find the best possible set of images to reconstruct the 3D structure. The structural studies in this thesis, exploit the advantages of single particle methods. 

The large data set generated by the SPI study of PR772 provides better statistics about the sample quality due to the use of GDVN, a container-free sample delivery method. By analyzing the diffusion map, we see that the use of GDVNs as a sample delivery method produces wide range of particle sizes owing to the large droplet that are created. 

The high-resolution structure of bacteriophage PR772 confirmed the speculation about the heteropentameric nature of the penton and revealed the new architecture of the vertex complex consisting of a hetero-pentameric penton formed with three copies of P5 and two copies of P31. The beta propeller region of P2, formed by domains I and II is bound to the N-terminal domain of P5. The structure also reveals new conformations of N-terminal and C-terminal region of P3 which play an important role in particle assembly and structural stability. 

The study of Melbournevirus revealed the protein composition in a packed particle. The CryoEM structure of Melbournevirus reveals a T=309 capsid with an inner lipid membrane. A dense body was found in the viral particle, a feature not observed in other viruses of the Marseilleviridae family. The density of this body is similar to a nucleic acid-protein complex. This observation, along with the histone-like protein identified during study, suggest genome organization in the viral particle, similar to higher organisms.

The soft X-ray microscope operated in the water-window shows the progression of the Cedratvirus lurbo infection in the host cell without the use of chemical fixation, staining, sample dehydration or polymer embedding. The study revealed a significant bioconversion from the host cell to the viral particle at later stages of infection.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 72
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1847
Keywords
PR772, phage, PRD1, Bacteriophage, coliphage, Melbournevirus, Cedratvirus, lurbo, Pithovirus, CryoEM, Single particle imaging, Coherent, Diffractive, Imaging, Soft X-ray, Microscopy, Microscope, GDVN, High resolution, XFEL, aerosol, Injection, electrospray, gas dynamic virtual nozzle, CDI, CXI, FEL
National Category
Structural Biology Biophysics
Research subject
Chemistry with specialization in Biophysics
Identifiers
urn:nbn:se:uu:diva-391671 (URN)978-91-513-0732-9 (ISBN)
Public defence
2019-10-11, Room C2:301, BMC, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2019-09-20 Created: 2019-08-25 Last updated: 2019-10-15

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Reddy, Hemanth K.N.Hajdu, JSvenda, Martin

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