DNA Packaging-Associated Hyper-Capsid Expansion of Bacteriophage T3

Serwer, Philip; Wright, Elena T.; Hakala, Kevin; Weintraub, Susan T.; Su, Min; Jiang, Wen

doi:10.1016/j.jmb.2010.01.058

Cited by 17 publications

(43 citation statements)

References 49 publications

(83 reference statements)

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“…The fact that all the particles were captured on the same TEM grid and imaged simultaneously in the same dataset eliminates the possibility of size variance caused by TEM magnification fluctuation. The molecular mechanism of this newly observed size reduction from CII to mature phage and its potential association with T7 DNA packaging (Serwer et al, 2010), remain to be elucidated. All other structural features, including the elliptical hexamers of CI, the L-shaped ridge of individual gp10 subunits, and the nearly equally-spaced concentric rings for genomic DNA of the mature phage (Fig.…”

Section: Resultsmentioning

confidence: 99%

Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies

Vago

Zhang

et al. 2014

Journal of Structural Biology

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Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (nonpurified His-tagged bacteriophage T7, His-tagged E. coli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures.

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Section: Resultsmentioning

confidence: 99%

Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies

Vago

Zhang

et al. 2014

Journal of Structural Biology

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“…, after the procapsid to mature capsid transition [45]. This second expansion is a hyper-expansion in that the shell becomes larger than the mature phage shell.…”

Section: Nucleic Acid Packaging Motorsmentioning

confidence: 99%

Proposed Ancestors of Phage Nucleic Acid Packaging Motors (and Cells)

Serwer

2011

Viruses

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I present a hypothesis that begins with the proposal that abiotic ancestors of phage RNA and DNA packaging systems (and cells) include mobile shells with an internal, molecule-transporting cavity. The foundations of this hypothesis include the conjecture that current nucleic acid packaging systems have imprints from abiotic ancestors. The abiotic shells (1) initially imbibe and later also bind and transport organic molecules, thereby providing a means for producing molecular interactions that are links in the chain of events that produces ancestors to the first molecules that are both information carrying and enzymatically active, and (2) are subsequently scaffolds on which proteins assemble to form ancestors common to both shells of viral capsids and cell membranes. Emergence of cells occurs via aggregation and merger of shells and internal contents. The hypothesis continues by using proposed imprints of abiotic and biotic ancestors to deduce an ancestral thermal ratchet-based DNA packaging motor that subsequently evolves to integrate a DNA packaging ATPase that provides a power stroke.

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“…Based on the lengths of the cleavage products, cleavage positions are sometimes quantized, as though occurring during the type 1 cycle stalls that trigger the type 2 cycle [30]. In further support of the proposed cleavage-promoting alternative pathway, artificial slowing of in vitro T3 packaging accelerates the maturation cleavage of concatemers [73].…”

Section: The Type 2 Cycle and Terminase Cleavage Of A Concatemermentioning

confidence: 99%

A Hypothesis for Bacteriophage DNA Packaging Motors

Serwer

2010

Viruses

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The hypothesis is presented that bacteriophage DNA packaging motors have a cycle comprised of bind/release thermal ratcheting with release-associated DNA pushing via ATP-dependent protein folding. The proposed protein folding occurs in crystallographically observed peptide segments that project into an axial channel of a protein 12-mer (connector) that serves, together with a coaxial ATPase multimer, as the entry portal. The proposed cycle begins when reverse thermal motion causes the connector’s peptide segments to signal the ATPase multimer to bind both ATP and the DNA molecule, thereby producing a dwell phase recently demonstrated by single-molecule procedures. The connector-associated peptide segments activate by transfer of energy from ATP during the dwell. The proposed function of connector/ATPase symmetry mismatches is to reduce thermal noise-induced signaling errors. After a dwell, ATP is cleaved and the DNA molecule released. The activated peptide segments push the released DNA molecule, thereby producing a burst phase recently shown to consist of four mini-bursts. The constraint of four mini-bursts is met by proposing that each mini-burst occurs via pushing by three of the 12 subunits of the connector. If all four mini-bursts occur, the cycle repeats. If the mini-bursts are not completed, a second cycle is superimposed on the first cycle. The existence of the second cycle is based on data recently obtained with bacteriophage T3. When both cycles stall, energy is diverted to expose the DNA molecule to maturation cleavage.

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DNA Packaging-Associated Hyper-Capsid Expansion of Bacteriophage T3

Cited by 17 publications

References 49 publications

Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies

Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies

Proposed Ancestors of Phage Nucleic Acid Packaging Motors (and Cells)

A Hypothesis for Bacteriophage DNA Packaging Motors

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