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- EMDB-26858: Pseudomonas phage E217 small terminase (TerS) -

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Basic information

Entry
Database: EMDB / ID: EMD-26858
TitlePseudomonas phage E217 small terminase (TerS)
Map data
Sample
  • Complex: Small terminase decamer from Pseudomonas phage E217
    • Protein or peptide: Small terminase
Keywordsphage small Terminase / decamer / oligomer / DNA BINDING PROTEIN
Function / homologyUncharacterized protein
Function and homology information
Biological speciesPseudomonas phage vB_PaeM_E217 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.38 Å
AuthorsLokareddy RK / Hou C-FD / Doll SG / Li F / Gillilan R / Forti F / Briani F / Cingolani G
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)GM100888 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)GM140733-01 United States
CitationJournal: J Mol Biol / Year: 2022
Title: Terminase Subunits from the Pseudomonas-Phage E217.
Authors: Ravi K Lokareddy / Chun-Feng David Hou / Steven G Doll / Fenglin Li / Richard E Gillilan / Francesca Forti / David S Horner / Federica Briani / Gino Cingolani /
Abstract: Pseudomonas phages are increasingly important biomedicines for phage therapy, but little is known about how these viruses package DNA. This paper explores the terminase subunits from the Myoviridae ...Pseudomonas phages are increasingly important biomedicines for phage therapy, but little is known about how these viruses package DNA. This paper explores the terminase subunits from the Myoviridae E217, a Pseudomonas-phage used in an experimental cocktail to eradicate P. aeruginosa in vitro and in animal models. We identified the large (TerL) and small (TerS) terminase subunits in two genes ∼58 kbs away from each other in the E217 genome. TerL presents a classical two-domain architecture, consisting of an N-terminal ATPase and C-terminal nuclease domain arranged into a bean-shaped tertiary structure. A 2.05 Å crystal structure of the C-terminal domain revealed an RNase H-like fold with two magnesium ions in the nuclease active site. Mutations in TerL residues involved in magnesium coordination had a dominant-negative effect on phage growth. However, the two ions identified in the active site were too far from each other to promote two-metal-ion catalysis, suggesting a conformational change is required for nuclease activity. We also determined a 3.38 Å cryo-EM reconstruction of E217 TerS that revealed a ring-like decamer, departing from the most common nonameric quaternary structure observed thus far. E217 TerS contains both N-terminal helix-turn-helix motifs enriched in basic residues and a central channel lined with basic residues large enough to accommodate double-stranded DNA. Overexpression of TerS caused a more than a 4-fold reduction of E217 burst size, suggesting a catalytic amount of the protein is required for packaging. Together, these data expand the molecular repertoire of viral terminase subunits to Pseudomonas-phages used for phage therapy.
History
DepositionMay 5, 2022-
Header (metadata) releaseSep 28, 2022-
Map releaseSep 28, 2022-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26858.png

Downloads & links

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Map

FileDownload / File: emd_26858.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.95 Å
Density
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-3.8016644 - 4.9353037
Average (Standard dev.)0.0040890733 (±0.19562541)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 190.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_26858_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_26858_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Small terminase decamer from Pseudomonas phage E217

EntireName: Small terminase decamer from Pseudomonas phage E217
Components
  • Complex: Small terminase decamer from Pseudomonas phage E217
    • Protein or peptide: Small terminase

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Supramolecule #1: Small terminase decamer from Pseudomonas phage E217

SupramoleculeName: Small terminase decamer from Pseudomonas phage E217 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pseudomonas phage vB_PaeM_E217 (virus)
Molecular weightTheoretical: 210 KDa

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Macromolecule #1: Small terminase

MacromoleculeName: Small terminase / type: protein_or_peptide / ID: 1 / Number of copies: 10 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas phage vB_PaeM_E217 (virus)
Molecular weightTheoretical: 21.30468 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MTKFYSPDDL VTPQEFADPH FAAINQKRFD LYIDLRVQGY SSWRVFRAIW GEEHMDGPAQ ARIFAMESNP YYRKQFKAKL NATKTSDLW NPKTALHELL QMVRDPTVKD SSRLSAIKEL NVLAEITFVD ESGKTRIGRG LADFYASEAE AQTATVAAAA E ANSYVPEG EEGDFPSPTP EPTEEDRANP I

UniProtKB: Uncharacterized protein

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm

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Image processing

Particle selectionNumber selected: 3500000
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C10 (10 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 587000
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-7uxe:
Pseudomonas phage E217 small terminase (TerS)

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