Biphasic Packing of DNA and Internal Proteins in Bacteriophage T4 Heads Revealed by Bubblegram Imaging

The virions of tailed bacteriophages and the evolutionarily related herpesviruses contain, in addition to highly condensed DNA, substantial quantities of internal proteins. These proteins (“ejection proteins”) have roles in scaffolding, maturational proteolysis, and cell-to-cell delivery. Whereas capsids are amenable to analysis at high resolution by cryo-electron microscopy, internal proteins have proved difficult to localize. In this study, we investigated the distribution of internal proteins in T4 by bubblegram imaging. Prior work has shown that at suitably high electron doses, radiation damage generates bubbles of hydrogen gas in nucleoprotein specimens. Using DNA origami as a test specimen, we show that DNA does not bubble under these conditions; it follows that bubbles represent markers for proteins. The interior of the prolate T4 head, ~1000 Å long by ~750 Å wide, has a bubble-free zone that is ~100–110 Å thick, underlying the capsid shell from which proteins are excluded by highly ordered DNA. Inside this zone, which is plausibly occupied by ~4 layers of coaxial spool, bubbles are generated at random locations in a disordered ensemble of internal proteins and the remainder of the genome.

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A resolution record for cryoEM

Faculty Reviews ◽

10.12703/r-01-000002 ◽

2021 ◽

Vol 10 ◽

Author(s):

Jonathan Ashmore ◽

Bridget Carragher ◽

Peter B Rosenthal ◽

William Weis

Keyword(s):

Electron Microscopy ◽

Image Analysis ◽

Structure Determination ◽

Test Specimen ◽

Structural Information ◽

Atomic Resolution ◽

Fast Growing ◽

Cryo Electron Microscopy ◽

New Instrument ◽

Resolution Structure

Cryo electron microscopy (cryoEM) is a fast-growing technique for structure determination. Two recent papers report the first atomic resolution structure of a protein obtained by averaging images of frozen-hydrated biomolecules. They both describe maps of symmetric apoferritin assemblies, a common test specimen, in unprecedented detail. New instrument improvements, different in the two studies, have contributed better images, and image analysis can extract structural information sufficient to resolve individual atomic positions. While true atomic resolution maps will not be routine for most proteins, the studies suggest structures determined by cryoEM will continue to improve, increasing their impact on biology and medicine.

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Structural and mechanistic bases for a potent HIV-1 capsid inhibitor

Science ◽

10.1126/science.abb4808 ◽

2020 ◽

Vol 370 (6514) ◽

pp. 360-364 ◽

Cited By ~ 10

Author(s):

Stephanie M. Bester ◽

Guochao Wei ◽

Haiyan Zhao ◽

Daniel Adu-Ampratwum ◽

Naseer Iqbal ◽

...

Keyword(s):

Principal Component ◽

X Ray ◽

X Ray Crystallography ◽

Cryo Electron Microscopy ◽

Capsid Shell ◽

Hydrogen Deuterium ◽

Infected Cells ◽

Long Acting ◽

Structural Insights ◽

Hiv 1

The potent HIV-1 capsid inhibitor GS-6207 is an investigational principal component of long-acting antiretroviral therapy. We found that GS-6207 inhibits HIV-1 by stabilizing and thereby preventing functional disassembly of the capsid shell in infected cells. X-ray crystallography, cryo–electron microscopy, and hydrogen-deuterium exchange experiments revealed that GS-6207 tightly binds two adjoining capsid subunits and promotes distal intra- and inter-hexamer interactions that stabilize the curved capsid lattice. In addition, GS-6207 interferes with capsid binding to the cellular HIV-1 cofactors Nup153 and CPSF6 that mediate viral nuclear import and direct integration into gene-rich regions of chromatin. These findings elucidate structural insights into the multimodal, potent antiviral activity of GS-6207 and provide a means for rationally developing second-generation therapies.

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Cryo-electron microscopy study of bacteriophage T4 displaying anthrax toxin proteins

Virology ◽

10.1016/j.virol.2007.05.036 ◽

2007 ◽

Vol 367 (2) ◽

pp. 422-427 ◽

Cited By ~ 12

Author(s):

Andrei Fokine ◽

Valorie D. Bowman ◽

Anthony J. Battisti ◽

Qin Li ◽

Paul R. Chipman ◽

...

Keyword(s):

Electron Microscopy ◽

Bacteriophage T4 ◽

Microscopy Study ◽

Electron Microscopy Study ◽

Anthrax Toxin ◽

Cryo Electron Microscopy

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Structural Analysis of Self-assembled Nanotubes of Bacteriophage T4 Capsid Protein gp23 by Cryo Electron Microscopy and Mass Spectrometry

Microscopy and Microanalysis ◽

10.1017/s1431927606066864 ◽

2006 ◽

Vol 12 (S02) ◽

pp. 658-659

Author(s):

BK Kaletas ◽

E Van Duijn ◽

AJ R Heck ◽

RB J Geels ◽

F De Haas ◽

...

Keyword(s):

Electron Microscopy ◽

Mass Spectrometry ◽

Structural Analysis ◽

Capsid Protein ◽

Bacteriophage T4 ◽

Cryo Electron Microscopy ◽

Self Assembled

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2006

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A nanoscale reciprocating rotary mechanism with coordinated mobility control

Nature Communications ◽

10.1038/s41467-021-27230-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Eva Bertosin ◽

Christopher M. Maffeo ◽

Thomas Drexler ◽

Maximilian N. Honemann ◽

Aleksei Aksimentiev ◽

...

Keyword(s):

Molecular Motors ◽

Large Scale ◽

Mechanical Deformation ◽

Dna Origami ◽

Mobility Control ◽

Mechanical Stiffness ◽

Allosteric Coupling ◽

Brownian Rotation ◽

Cryo Electron Microscopy ◽

Dynamics Simulations

AbstractBiological molecular motors transform chemical energy into mechanical work by coupling cyclic catalytic reactions to large-scale structural transitions. Mechanical deformation can be surprisingly efficient in realizing such coupling, as demonstrated by the F1FO ATP synthase. Here, we describe a synthetic molecular mechanism that transforms a rotary motion of an asymmetric camshaft into reciprocating large-scale transitions in a surrounding stator orchestrated by mechanical deformation. We design the mechanism using DNA origami, characterize its structure via cryo-electron microscopy, and examine its dynamic behavior using single-particle fluorescence microscopy and molecular dynamics simulations. While the camshaft can rotate inside the stator by diffusion, the stator’s mechanics makes the camshaft pause at preferred orientations. By changing the stator’s mechanical stiffness, we accelerate or suppress the Brownian rotation, demonstrating an allosteric coupling between the camshaft and the stator. Our mechanism provides a framework for manufacturing artificial nanomachines that function because of coordinated movements of their components.

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Cryo-Electron Microscopy of Highly Condensed DNA inside Virus Capsids

Microscopy and Microanalysis ◽

10.1017/s143192760550713x ◽

2005 ◽

Vol 11 (S02) ◽

Author(s):

A C Steven ◽

N Cheng ◽

B Mai ◽

A Jones ◽

C Butan ◽

...

Keyword(s):

Electron Microscopy ◽

Cryo Electron Microscopy ◽

Virus Capsids ◽

Condensed Dna

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Capsid structure of channel catfish virus, an evolutionarily distant herpesvirus, by cryo-Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010016830x ◽

1994 ◽

Vol 52 ◽

pp. 114-115

Author(s):

Benes L. Trus ◽

Andrew J. Davison ◽

Frank P. Booy ◽

Alasdair C. Steven

Keyword(s):

Electron Microscopy ◽

Channel Catfish ◽

3 Dimensional ◽

Channel Catfish Virus ◽

Cryo Electron Microscopy ◽

Capsid Shell ◽

Wide Range ◽

Dimensional Reconstruction ◽

Molecular Anatomy ◽

Evident Relationship

Herpesviruses comprise an extensive family of enveloped DNA-containing animal viruses. Although they infect a wide range of vertebrate hosts and their linear double-stranded genomes vary substantially in size and other properties, the nucleocapsid appears to be a conservative element of viral design. The capsid shell is icosahedrally symmetric (T=16), and in the case of alphaherpesviruses is 125 nm in diameter and 15nm thick. Recently, we have studied the molecular anatomy of herpes simplex virus1 (HSV-1), whose capsid contains four major proteins, by combining cryo-electron microscopy and 3-dimensional reconstruction with biochemical depletion experiments and antibody-labelling. In order to probe structural perturbations attributable to evolutionary differences, we have extended these studies to channel catfish virus. CCV exhibits the gross morphology of a herpesvirus, although no evident relationship to other herpesviruses was found in an analysis of proteins predicted from its complete DNA sequence.

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Structural basis of σ appropriation

Nucleic Acids Research ◽

10.1093/nar/gkz682 ◽

2019 ◽

Vol 47 (17) ◽

pp. 9423-9432 ◽

Cited By ~ 6

Author(s):

Jing Shi ◽

Aijia Wen ◽

Minxing Zhao ◽

Linlin You ◽

Yu Zhang ◽

...

Keyword(s):

Electron Microscopy ◽

Transcription Factors ◽

Structural Information ◽

Bacteriophage T4 ◽

Transcription Activation ◽

Structural Basis ◽

Concerted Effort ◽

Double Stranded Dna ◽

Cryo Electron Microscopy ◽

Large Conformational Change

Abstract Bacteriophage T4 middle promoters are activated through a process called σ appropriation, which requires the concerted effort of two T4-encoded transcription factors: AsiA and MotA. Despite extensive biochemical and genetic analyses, puzzle remains, in part, because of a lack of precise structural information for σ appropriation complex. Here, we report a single-particle cryo-electron microscopy (cryo-EM) structure of an intact σ appropriation complex, comprising AsiA, MotA, Escherichia coli RNA polymerase (RNAP), σ70 and a T4 middle promoter. As expected, AsiA binds to and remodels σ region 4 to prevent its contact with host promoters. Unexpectedly, AsiA undergoes a large conformational change, takes over the job of σ region 4 and provides an anchor point for the upstream double-stranded DNA. Because σ region 4 is conserved among bacteria, other transcription factors may use the same strategy to alter the landscape of transcription immediately. Together, the structure provides a foundation for understanding σ appropriation and transcription activation.

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The Architecture and Chemical Stability of the Archaeal Sulfolobus Turreted Icosahedral Virus

Journal of Virology ◽

10.1128/jvi.00708-10 ◽

2010 ◽

Vol 84 (18) ◽

pp. 9575-9583 ◽

Cited By ~ 27

Author(s):

Reza Khayat ◽

Chi-yu Fu ◽

Alice C. Ortmann ◽

Mark J. Young ◽

John E. Johnson

Keyword(s):

Major Capsid Protein ◽

Virus Genome ◽

Archaeal Virus ◽

Double Stranded Dna ◽

Cryo Electron Microscopy ◽

Resolution Electron ◽

Capsid Shell ◽

Icosahedral Virus ◽

Sulfolobus Turreted Icosahedral Virus ◽

Made In

ABSTRACT Viruses utilize a diverse array of mechanisms to deliver their genomes into hosts. While great strides have been made in understanding the genome delivery of eukaryotic and prokaryotic viruses, little is known about archaeal virus genome delivery and the associated particle changes. The Sulfolobus turreted icosahedral virus (STIV) is a double-stranded DNA (dsDNA) archaeal virus that contains a host-derived membrane sandwiched between the genome and the proteinaceous capsid shell. Using cryo-electron microscopy (cryo-EM) and different biochemical treatments, we identified three viral morphologies that may correspond to biochemical disassembly states of STIV. One of these morphologies was subtly different from the previously published 27-Å-resolution electron density that was interpreted with the crystal structure of the major capsid protein (MCP). However, these particles could be analyzed at 12.5-Å resolution by cryo-EM. Comparing these two structures, we identified the location of multiple proteins forming the large turret-like appendages at the icosahedral vertices, observed heterogeneous glycosylation of the capsid shell, and identified mobile MCP C-terminal arms responsible for tethering and releasing the underlying viral membrane to and from the capsid shell. Collectively, our studies allow us to propose a fusogenic mechanism of genome delivery by STIV, in which the dismantled capsid shell allows for the fusion of the viral and host membranes and the internalization of the viral genome.

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