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- PDB-6v1i: Cryo-EM reconstruction of the thermophilic bacteriophage P74-26 s... -

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Basic information

Entry
Database: PDB / ID: 6v1i
TitleCryo-EM reconstruction of the thermophilic bacteriophage P74-26 small terminase- symmetric
ComponentsSmall terminase protein
KeywordsVIRAL PROTEIN / small terminase / bacteriophage / helix-turn-helix / motor
Function / homologyUncharacterized protein
Function and homology information
Biological speciesThermus virus P74-26
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsHayes, J.A. / Hilbert, B.J. / Gaubitz, C. / Stone, N.P. / Kelch, B.A.
Funding support United States, 2items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)1817338 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1F31GM121019-01A1 United States
CitationJournal: J Biol Chem / Year: 2020
Title: A thermophilic phage uses a small terminase protein with a fixed helix-turn-helix geometry.
Authors: Janelle A Hayes / Brendan J Hilbert / Christl Gaubitz / Nicholas P Stone / Brian A Kelch /
Abstract: Tailed bacteriophages use a DNA-packaging motor to encapsulate their genome during viral particle assembly. The small terminase (TerS) component of this DNA-packaging machinery acts as a molecular ...Tailed bacteriophages use a DNA-packaging motor to encapsulate their genome during viral particle assembly. The small terminase (TerS) component of this DNA-packaging machinery acts as a molecular matchmaker that recognizes both the viral genome and the main motor component, the large terminase (TerL). However, how TerS binds DNA and the TerL protein remains unclear. Here we identified gp83 of the thermophilic bacteriophage P74-26 as the TerS protein. We found that TerS oligomerizes into a nonamer that binds DNA, stimulates TerL ATPase activity, and inhibits TerL nuclease activity. A cryo-EM structure of TerS revealed that it forms a ring with a wide central pore and radially arrayed helix-turn-helix domains. The structure further showed that these helix-turn-helix domains, which are thought to bind DNA by wrapping the double helix around the ring, are rigidly held in an orientation distinct from that seen in other TerS proteins. This rigid arrangement of the putative DNA-binding domain imposed strong constraints on how TerS can bind DNA. Finally, the TerS structure lacked the conserved C-terminal β-barrel domain used by other TerS proteins for binding TerL. This suggests that a well-ordered C-terminal β-barrel domain is not required for TerS to carry out its matchmaking function. Our work highlights a thermophilic system for studying the role of small terminase proteins in viral maturation and presents the structure of TerS, revealing key differences between this thermophilic phage and its mesophilic counterparts.
History
DepositionNov 20, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 12, 2020Provider: repository / Type: Initial release
Revision 1.1Feb 19, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Apr 1, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)171,7289
Polymers171,7289
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Small terminase protein


Mass: 19080.887 Da / Num. of mol.: 9
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus virus P74-26 / Gene: P74p83 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: A7XXR0

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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: Gene product 83 of the Thermus thermophilus bacteriophage P74-26
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.171 MDa / Experimental value: YES
Source (natural)Organism: Thermus virus P74-26
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium chlorideNaClSodium chloride1
220 mMTris(hydroxymethyl)aminomethane hydrochlorideTris-HClTris1
30.015 %A8-35A8-351
SpecimenConc.: 3.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse (PDI= 1.000)
Specimen supportDetails: 20 mA
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 13000 X / Calibrated defocus min: 1400 nm / Calibrated defocus max: 2600 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 2822

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1cisTEMparticle selection
4Gctf1.06CTF correction
7Coot0.8model fitting
9PHENIX1.13model refinement
10RELION3initial Euler assignmentBeta
11RELION3final Euler assignmentBeta
12RELION3classificationBeta
13RELION33D reconstructionBeta
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C9 (9 fold cyclic)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 84860 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 4XVN

4xvn


Pdb chain-ID: A / Accession code: 4XVN / Pdb chain residue range: 1-137 / Source name: PDB / Type: experimental model

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