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

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Basic information

Entry
Database: PDB / ID: 7uxe
TitlePseudomonas phage E217 small terminase (TerS)
ComponentsSmall terminase
KeywordsDNA BINDING PROTEIN / phage small Terminase / decamer / oligomer
Function / homologyUncharacterized protein
Function and homology information
Biological speciesPseudomonas phage vB_PaeM_E217 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.38 Å
AuthorsLokareddy, R.K. / Hou, C.-F.D. / Doll, S.G. / Li, F. / Gillilan, R. / Forti, F. / Briani, F. / Cingolani, G.
Funding support United States, 2items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 28, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Small terminase
B: Small terminase
C: Small terminase
D: Small terminase
E: Small terminase
F: Small terminase
G: Small terminase
H: Small terminase
I: Small terminase
J: Small terminase


Theoretical massNumber of molelcules
Total (without water)213,04710
Polymers213,04710
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Small terminase


Mass: 21304.680 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas phage vB_PaeM_E217 (virus) / Gene: vBPaeME217_00078 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A2K8I4H6

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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: Small terminase decamer from Pseudomonas phage E217 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.21 MDa / Experimental value: NO
Source (natural)Organism: Pseudomonas phage vB_PaeM_E217 (virus)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
4cryoSPARC3.3.2CTF correction
7UCSF Chimera1.3model fitting
12cryoSPARC3.3.23D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3500000
SymmetryPoint symmetry: C10 (10 fold cyclic)
3D reconstructionResolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 587000 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0049720
ELECTRON MICROSCOPYf_angle_d0.7513140
ELECTRON MICROSCOPYf_dihedral_angle_d5.7841260
ELECTRON MICROSCOPYf_chiral_restr0.0391390
ELECTRON MICROSCOPYf_plane_restr0.0051690

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