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- PDB-6ysz: Cryo-EM structure of T7 bacteriophage DNA translocation gp15 core... -

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Entry
Database: PDB / ID: 6ysz
TitleCryo-EM structure of T7 bacteriophage DNA translocation gp15 core protein intermediate assembly
ComponentsInternal virion protein gp15
KeywordsVIRAL PROTEIN / DNA TRANSLOCATION / VIRAL INFECTION / PERIPLASMIC SPACE COMPLEX.
Function / homologyInternal virion protein Gp15 / host cell periplasmic space / symbiont genome ejection through host cell envelope, short tail mechanism / virion component / Internal virion protein gp15
Function and homology information
Biological speciesEscherichia phage T7 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsPerez-Ruiz, M. / Pulido-Cid, M. / Luque-Ortega, J.R. / Cuervo, A. / Carrascosa, J.L.
Funding support Spain, 3items
OrganizationGrant numberCountry
Spanish Ministry of Science, Innovation, and UniversitiesBFU 2014-54181 Spain
Spanish Ministry of Science, Innovation, and UniversitiesSEV-2013-0347 Spain
Spanish Ministry of Science, Innovation, and UniversitiesBES-2015-073615 Spain
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Assisted assembly of bacteriophage T7 core components for genome translocation across the bacterial envelope.
Authors: Mar Pérez-Ruiz / Mar Pulido-Cid / Juan Román Luque-Ortega / José María Valpuesta / Ana Cuervo / José L Carrascosa /
Abstract: In most bacteriophages, genome transport across bacterial envelopes is carried out by the tail machinery. In viruses of the family, in which the tail is not long enough to traverse the bacterial ...In most bacteriophages, genome transport across bacterial envelopes is carried out by the tail machinery. In viruses of the family, in which the tail is not long enough to traverse the bacterial wall, it has been postulated that viral core proteins assembled inside the viral head are translocated and reassembled into a tube within the periplasm that extends the tail channel. Bacteriophage T7 infects , and despite extensive studies, the precise mechanism by which its genome is translocated remains unknown. Using cryo-electron microscopy, we have resolved the structure of two different assemblies of the T7 DNA translocation complex composed of the core proteins gp15 and gp16. Gp15 alone forms a partially folded hexamer, which is further assembled upon interaction with gp16 into a tubular structure, forming a channel that could allow DNA passage. The structure of the gp15-gp16 complex also shows the location within gp16 of a canonical transglycosylase motif involved in the degradation of the bacterial peptidoglycan layer. This complex docks well in the tail extension structure found in the periplasm of T7-infected bacteria and matches the sixfold symmetry of the phage tail. In such cases, gp15 and gp16 that are initially present in the T7 capsid eightfold-symmetric core would change their oligomeric state upon reassembly in the periplasm. Altogether, these results allow us to propose a model for the assembly of the core translocation complex in the periplasm, which furthers understanding of the molecular mechanism involved in the release of T7 viral DNA into the bacterial cytoplasm.
History
DepositionApr 23, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 8, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Internal virion protein gp15
B: Internal virion protein gp15
C: Internal virion protein gp15
D: Internal virion protein gp15
E: Internal virion protein gp15
F: Internal virion protein gp15


Theoretical massNumber of molelcules
Total (without water)530,2636
Polymers530,2636
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: equilibrium centrifugation, light scattering
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area28690 Å2
ΔGint-34 kcal/mol
Surface area84550 Å2
MethodPISA

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Components

#1: Protein
Internal virion protein gp15 / Gene product 15 / Gp15


Mass: 88377.219 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage T7 (virus) / Gene: 15 / Production host: Escherichia coli (E. coli) / References: UniProt: P03725

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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: gp15 tubular core protein / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.543 MDa / Experimental value: YES
Source (natural)Organism: Escherichia phage T7 (virus)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: C41
Buffer solutionpH: 8
Buffer component
IDConc.FormulaBuffer-ID
1100 mMTris1
2100 mMNaCitrate1
310 mMMgCl21
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 293.15 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 120000 X / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 35 sec. / Electron dose: 30 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2470
Image scansMovie frames/image: 32

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Processing

EM software
IDNameCategory
1Gautomatchparticle selection
2EPUimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50980 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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