Campbell, Patricia L
(2019)
Assembly and Structure of Bacteriophage Capsids and Tails.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Phages HK97 and λ are model systems that have aided in the understanding of capsid and tail assembly. HK97 capsid assembly involves the formation of a T=7 icosahedral lattice from 415 monomers of the major capsid protein (mcp), 12 monomers of portal protein, and ~120 copies of the protease. Salt bridges have been found to play a critical role in the assembly pathway, but it is not clear how capsid size is determined. Phage phi1026b is larger than HK97 but the mcp shares 50% sequence similarity with HK97. We hypothesize that comparing the atomic models of phi1026b and HK97 will help illuminate conserved and unique interactions that may play a role in assembly and potentially size determination. By utilizing the FEI Krios microscope, Falcon II camera, and EPU data acquisition software, high-resolution structures were generated which allowed for the generation of atomic models of the phi1026b Prohead I and Head capsids. Differences were observed in the organization of the delta domain, capsomers, and intra-capsomer interactions. The E153-R210 salt bridge that is maintained throughout HK97 capsid assembly is not present between two monomers in the phi1206b Prohead I structure. Phage λ tail assembly proceeds in a stepwise manner in which tail proteins form an initiator complex onto which the major tail tube protein, gpV, polymerizes and is terminated by the tail termination protein, gpU. The tube domain of gpV has been studied in its monomeric form using NMR but the in vivo form has not been visualized. By generating a subnanometer structure for the λ tail tube, we are able to show how gpV undergoes structural changes to facilitate assembly. Also, a putative initiator complex comprised of the tail proteins gpG, gpT, gpH, gpM, gpL, gpI, and gpJ was isolated and characterized using microscopy. Finally, a subnanometer structure for the λ tail tip complex (TTC) is presented providing some initial insight into the organization of the λ TTC. Overall, this research intends to optimize structure determination with the cryo-EM, thereby facilitating studies on the structure and assembly of large viruses.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
| Creators | Email | Pitt Username | ORCID  |
|---|
| Campbell, Patricia L | plc25@pitt.edu | plc25 | |
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| ETD Committee: |
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| Date: |
18 September 2019 |
| Date Type: |
Publication |
| Defense Date: |
25 July 2019 |
| Approval Date: |
18 September 2019 |
| Submission Date: |
1 August 2019 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
195 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Molecular Biophysics and Structural Biology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Bacteriophage
Capsid
Cryo-EM
Structural Biology
Tail
Tail tip complex |
| Date Deposited: |
18 Sep 2019 14:07 |
| Last Modified: |
18 Sep 2019 14:07 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/37268 |
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