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- PDB-3j4b: Structure of T7 gatekeeper protein (gp11) -

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Basic information

Entry
Database: PDB / ID: 3j4b
TitleStructure of T7 gatekeeper protein (gp11)
ComponentsTail tubular protein A
KeywordsVIRAL PROTEIN / Bacteriophage / DNA ejection / tail complex / gatekeeper
Function / homologyTail tubular protein Gp11 / Tail tubular protein / virus tail, tube / symbiont genome ejection through host cell envelope, short tail mechanism / Tail tubular protein gp11
Function and homology information
Biological speciesEnterobacteria phage T7 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 12 Å
AuthorsCuervo, A. / Pulido-Cid, M. / Chagoyen, M. / Arranz, R. / Gonzalez-Garcia, V.A. / Garcia-Doval, C. / Caston, J.R. / Valpuesta, J.M. / van Raaij, M.J. / Martin-Benito, J. / Carrascosa, J.L.
CitationJournal: J Biol Chem / Year: 2013
Title: Structural characterization of the bacteriophage T7 tail machinery.
Authors: Ana Cuervo / Mar Pulido-Cid / Mónica Chagoyen / Rocío Arranz / Verónica A González-García / Carmela Garcia-Doval / José R Castón / José M Valpuesta / Mark J van Raaij / Jaime Martín- ...Authors: Ana Cuervo / Mar Pulido-Cid / Mónica Chagoyen / Rocío Arranz / Verónica A González-García / Carmela Garcia-Doval / José R Castón / José M Valpuesta / Mark J van Raaij / Jaime Martín-Benito / José L Carrascosa /
Abstract: Most bacterial viruses need a specialized machinery, called "tail," to inject their genomes inside the bacterial cytoplasm without disrupting the cellular integrity. Bacteriophage T7 is a well ...Most bacterial viruses need a specialized machinery, called "tail," to inject their genomes inside the bacterial cytoplasm without disrupting the cellular integrity. Bacteriophage T7 is a well characterized member of the Podoviridae family infecting Escherichia coli, and it has a short noncontractile tail that assembles sequentially on the viral head after DNA packaging. The T7 tail is a complex of around 2.7 MDa composed of at least four proteins as follows: the connector (gene product 8, gp8), the tail tubular proteins gp11 and gp12, and the fibers (gp17). Using cryo-electron microscopy and single particle image reconstruction techniques, we have determined the precise topology of the tail proteins by comparing the structure of the T7 tail extracted from viruses and a complex formed by recombinant gp8, gp11, and gp12 proteins. Furthermore, the order of assembly of the structural components within the complex was deduced from interaction assays with cloned and purified tail proteins. The existence of common folds among similar tail proteins allowed us to obtain pseudo-atomic threaded models of gp8 (connector) and gp11 (gatekeeper) proteins, which were docked into the corresponding cryo-EM volumes of the tail complex. This pseudo-atomic model of the connector-gatekeeper interaction revealed the existence of a common molecular architecture among viruses belonging to the three tailed bacteriophage families, strongly suggesting that a common molecular mechanism has been favored during evolution to coordinate the transition between DNA packaging and tail assembly.
History
DepositionJul 9, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 7, 2013Provider: repository / Type: Initial release
Revision 1.1Sep 25, 2013Group: Database references
Revision 1.2Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id / _em_software.name
Revision 1.3Feb 21, 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: Tail tubular protein A
J: Tail tubular protein A
K: Tail tubular protein A
L: Tail tubular protein A
B: Tail tubular protein A
C: Tail tubular protein A
D: Tail tubular protein A
E: Tail tubular protein A
F: Tail tubular protein A
G: Tail tubular protein A
H: Tail tubular protein A
I: Tail tubular protein A


Theoretical massNumber of molelcules
Total (without water)251,24512
Polymers251,24512
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Tail tubular protein A / gp11 / gatekeeper / Coordinate model: Cα atoms only


Mass: 20937.119 Da / Num. of mol.: 12 / Fragment: UNP residues 13-196
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacteria phage T7 (virus) / Gene: 11 / Plasmid: pRSET-B / Production host: Escherichia coli (E. coli) / Strain (production host): C41 / References: UniProt: P03746

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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: T7 tail complex formed by proteins gp8, gp11 and gp12 / Type: VIRUS / Details: gp8 (12mer), gp11 (12mer), gp12 (6mer)
Molecular weightValue: 1.5 MDa / Experimental value: NO
Details of virusHost category: BACTERIA / Type: VIRION
Natural hostOrganism: Escherichia coli
Buffer solutionName: 50 mM Tris-HCl, 10 mM MgCl2, 100 mM NaCl / pH: 7.8 / Details: 50 mM Tris-HCl, 10 mM MgCl2, 100 mM NaCl
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: R2/2 Quantifoil coated with a thin carbon layer
VitrificationInstrument: LEICA EM CPC / Cryogen name: ETHANE
Details: Samples were applied to grids for 1 minute, blotted and plunged into liquid ethane (LEICA EM CPC).
Method: Samples were applied to grids for 1 minute, blotted and plunged into liquid ethane.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Nov 8, 2012
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 108696 X / Calibrated magnification: 108696 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Cs: 2.26 mm / Camera length: 0 mm
Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN / Temperature (max): 113 K / Temperature (min): 91 K / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 10 e/Å2 / Film or detector model: FEI EAGLE (4k x 4k)
Image scansNum. digital images: 264
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1UCSF Chimeramodel fitting
2EMAN3D reconstruction
3SPIDER3D reconstruction
4Xmipp3D reconstruction
CTF correctionDetails: each micrograph
SymmetryPoint symmetry: C12 (12 fold cyclic)
3D reconstructionMethod: Cross-common lines / Resolution: 12 Å / Resolution method: FSC / Num. of particles: 1820 / Nominal pixel size: 2.75 Å / Actual pixel size: 2.75 Å / Details: (Single particle--Applied symmetry: C6) / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Volumetric correlation
Details: REFINEMENT PROTOCOL--rigid body DETAILS--One monomer was manually fitted and then the oligomer was generated using SITUS program
Atomic model buildingPDB-ID: 1VT0

1vt0
PDB Unreleased entry


Accession code: 1VT0 / Source name: PDB / Type: experimental model
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms2208 0 0 0 2208

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