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- PDB-8vxq: Cryo-EM structure of phage DEV ejection proteins gp72:gp73 -

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Basic information

Entry
Database: PDB / ID: 8vxq
TitleCryo-EM structure of phage DEV ejection proteins gp72:gp73
Components
  • N4 gp52-like protein
  • gp72
KeywordsVIRAL PROTEIN / phage / bacteriophage / STRUCTURAL PROTEIN / outer membrane protein / gp73 / gp74 / DEV
Function / homologyUncharacterized protein / N4 gp52-like protein
Function and homology information
Biological speciesPseudomonas phage vB_PaeP_DEV (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsIglesias, S.M. / Cingolani, G.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)GM140733 United States
Citation
Journal: Nat Commun / Year: 2024
Title: Integrative structural analysis of Pseudomonas phage DEV reveals a genome ejection motor.
Authors: Ravi K Lokareddy / Chun-Feng David Hou / Francesca Forti / Stephano M Iglesias / Fenglin Li / Mikhail Pavlenok / David S Horner / Michael Niederweis / Federica Briani / Gino Cingolani /
Abstract: DEV is an obligatory lytic Pseudomonas phage of the N4-like genus, recently reclassified as Schitoviridae. The DEV genome encodes 91 ORFs, including a 3398 amino acid virion-associated RNA polymerase ...DEV is an obligatory lytic Pseudomonas phage of the N4-like genus, recently reclassified as Schitoviridae. The DEV genome encodes 91 ORFs, including a 3398 amino acid virion-associated RNA polymerase (vRNAP). Here, we describe the complete architecture of DEV, determined using a combination of cryo-electron microscopy localized reconstruction, biochemical methods, and genetic knockouts. We built de novo structures of all capsid factors and tail components involved in host attachment. We demonstrate that DEV long tail fibers are essential for infection of Pseudomonas aeruginosa but dispensable for infecting mutants with a truncated lipopolysaccharide devoid of the O-antigen. We determine that DEV vRNAP is part of a three-gene operon conserved in 191 Schitoviridae genomes. We propose these three proteins are ejected into the host to form a genome ejection motor spanning the cell envelope. We posit that the design principles of the DEV ejection apparatus are conserved in all Schitoviridae.
#1: Journal: Res Sq / Year: 2024
Title: Integrative structural analysis of phage DEV reveals a genome ejection motor.
Authors: Gino Cingolani / Ravi Lokareddy / Chun-Feng Hou / Francesca Forti / Stephano Iglesias / Fenglin Li / Mikhail Pavlenok / Michael Niederweis / Federica Briani
Abstract: DEV is an obligatory lytic phage of the N4-like genus, recently reclassified as . The DEV genome encodes 91 ORFs, including a 3,398 amino acid virion-associated RNA polymerase. Here, we describe the ...DEV is an obligatory lytic phage of the N4-like genus, recently reclassified as . The DEV genome encodes 91 ORFs, including a 3,398 amino acid virion-associated RNA polymerase. Here, we describe the complete architecture of DEV, determined using a combination of cryo-electron microscopy localized reconstruction, biochemical methods, and genetic knockouts. We built de structures of all capsid factors and tail components involved in host attachment. We demonstrate that DEV long tail fibers are essential for infection of and dispensable for infecting mutants with a truncated lipopolysaccharide devoid of the O-antigen. We identified DEV ejection proteins and, unexpectedly, found that the giant DEV RNA polymerase, the hallmark of the family, is an ejection protein. We propose that DEV ejection proteins form a genome ejection motor across the host cell envelope and that these structural principles are conserved in all .
History
DepositionFeb 5, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 3, 2024Provider: repository / Type: Initial release
Revision 1.0Apr 3, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.0Apr 3, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Apr 3, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 3, 2024Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 3, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 3, 2024Data content type: Mask / Data content type: Mask / Provider: repository / Type: Initial release
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Revision 1.1Oct 16, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _em_admin.last_update
Revision 1.2May 14, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 14, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
R: gp72
J: gp72
K: gp72
L: gp72
M: gp72
N: gp72
O: gp72
P: gp72
Q: gp72
A: N4 gp52-like protein
B: N4 gp52-like protein
C: N4 gp52-like protein
D: N4 gp52-like protein
E: N4 gp52-like protein
F: N4 gp52-like protein
G: N4 gp52-like protein
H: N4 gp52-like protein
I: N4 gp52-like protein


Theoretical massNumber of molelcules
Total (without water)664,40418
Polymers664,40418
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
gp72


Mass: 57183.238 Da / Num. of mol.: 9 / Source method: isolated from a natural source / Source: (natural) Pseudomonas phage vB_PaeP_DEV (virus) / References: UniProt: A0A2K8HKQ8
#2: Protein
N4 gp52-like protein


Mass: 16639.414 Da / Num. of mol.: 9 / Source method: isolated from a natural source / Source: (natural) Pseudomonas phage vB_PaeP_DEV (virus) / References: UniProt: A0A2K8I0A4
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Pseudomonas phage vB_PaeP_DEV / Type: VIRUS / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Pseudomonas phage vB_PaeP_DEV (virus)
Details of virusEmpty: NO / Enveloped: NO / Isolate: OTHER / Type: VIRION
Natural hostOrganism: Pseudomonas aeruginosa
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 1 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C9 (9 fold cyclic)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 61482 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00531950
ELECTRON MICROSCOPYf_angle_d0.74442984
ELECTRON MICROSCOPYf_dihedral_angle_d3.8894581
ELECTRON MICROSCOPYf_chiral_restr0.0384644
ELECTRON MICROSCOPYf_plane_restr0.0046003

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