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- PDB-7k5c: Structure of T7 DNA ejectosome periplasmic tunnel -

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Basic information

Entry
Database: PDB / ID: 7k5c
TitleStructure of T7 DNA ejectosome periplasmic tunnel
Components
  • Internal virion protein gp15
  • Peptidoglycan transglycosylase gp16
KeywordsVIRAL PROTEIN / T7 / ejectosome / ejection protein / genome-delivery / podoviridae
Function / homology
Function and homology information


symbiont genome ejection through host cell envelope / host cell periplasmic space / : / symbiont entry into host cell via disruption of host cell wall peptidoglycan / lytic transglycosylase activity / symbiont genome ejection through host cell envelope, short tail mechanism / symbiont entry into host cell via disruption of host cell envelope / peptidoglycan metabolic process / symbiont entry into host / virion component ...symbiont genome ejection through host cell envelope / host cell periplasmic space / : / symbiont entry into host cell via disruption of host cell wall peptidoglycan / lytic transglycosylase activity / symbiont genome ejection through host cell envelope, short tail mechanism / symbiont entry into host cell via disruption of host cell envelope / peptidoglycan metabolic process / symbiont entry into host / virion component / killing of cells of another organism / hydrolase activity / defense response to bacterium / host cell plasma membrane / membrane
Similarity search - Function
Internal virion protein Gp16 / Internal virion protein Gp15 / Prokaryotic transglycosylase, active site / Prokaryotic transglycosylases signature. / Transglycosylase SLT domain 1 / Transglycosylase SLT domain / Lysozyme-like domain superfamily
Similarity search - Domain/homology
Internal virion protein gp15 / Peptidoglycan transglycosylase gp16
Similarity search - Component
Biological speciesEscherichia phage T7 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsSwanson, N. / Cingolani, G. / Pumroy, R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM100888 United States
CitationJournal: Mol Cell / Year: 2021
Title: Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution.
Authors: Nicholas A Swanson / Ravi K Lokareddy / Fenglin Li / Chun-Feng David Hou / Sebastian Leptihn / Mikhail Pavlenok / Michael Niederweis / Ruth A Pumroy / Vera Y Moiseenkova-Bell / Gino Cingolani /
Abstract: Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Seventy years later, our understanding of viral genome ...Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Seventy years later, our understanding of viral genome delivery in prokaryotes remains limited, especially for short-tailed phages of the Podoviridae family. These viruses expel mysterious ejection proteins found inside the capsid to form a DNA-ejectosome for genome delivery into bacteria. Here, we reconstitute the phage T7 DNA-ejectosome components gp14, gp15, and gp16 and solve the periplasmic tunnel structure at 2.7 Å resolution. We find that gp14 forms an outer membrane pore, gp15 assembles into a 210 Å hexameric DNA tube spanning the host periplasm, and gp16 extends into the host cytoplasm forming a ∼4,200 residue hub. Gp16 promotes gp15 oligomerization, coordinating peptidoglycan hydrolysis, DNA binding, and lipid insertion. The reconstituted gp15:gp16 complex lacks channel-forming activity, suggesting that the pore for DNA passage forms only transiently during genome ejection.
History
DepositionSep 16, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 28, 2021Provider: repository / Type: Initial release
Revision 1.1Aug 18, 2021Group: Database references / Category: citation / database_2
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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  • Deposited structure unit
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  • EMDB-22680
  • Imaged by UCSF Chimera
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Assembly

Deposited unit
H: Internal virion protein gp15
G: Peptidoglycan transglycosylase gp16
A: Internal virion protein gp15
B: Peptidoglycan transglycosylase gp16
C: Internal virion protein gp15
D: Peptidoglycan transglycosylase gp16
E: Internal virion protein gp15
F: Peptidoglycan transglycosylase gp16
I: Internal virion protein gp15
J: Peptidoglycan transglycosylase gp16
K: Internal virion protein gp15
L: Peptidoglycan transglycosylase gp16


Theoretical massNumber of molelcules
Total (without water)1,370,89312
Polymers1,370,89312
Non-polymers00
Water3,945219
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, equilibrium centrifugation
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area94700 Å2
ΔGint-292 kcal/mol
Surface area229440 Å2

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Components

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


Mass: 84454.008 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
#2: Protein
Peptidoglycan transglycosylase gp16 / Internal core protein gp16


Mass: 144028.219 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage T7 (virus) / Gene: 16 / Production host: Escherichia coli (E. coli)
References: UniProt: P03726, Lyases; Carbon-oxygen lyases; Acting on polysaccharides
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 219 / Source method: isolated from a natural source / Formula: H2O

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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: Multisubunit T7 DNA ejectosome periplasmic tunnel with 6x copies of Gp15 and 6x copies of Gp16
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.59 MDa
Source (natural)Organism: Escherichia phage T7 (virus)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8.5 / Details: Solutions were made fresh and filtered.
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMTris bufferTris/HCl1
210 mMmagnesium chlorideMgCl21
3100 mMsodium citrateNa3C6H5O71
SpecimenConc.: 2.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: This sample was monodisperse, but high concentration. Some preferred orientation.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 278 K / Details: blot for 6 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 2500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.4 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8601

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3.0-bparticle selectionManual and Auto-picking were completed with Relion
2LatitudeSimage acquisition
4RELION3.0-bCTF correction
7UCSF Chimera1.14model fitting
9RELION3.0-binitial Euler assignment
10RELION3.0-bfinal Euler assignment
11RELION3.0-bclassification
12RELION3.0-b3D reconstruction
13PHENIX1.17model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4700000
Details: Manually picked 2000 particles using Relion GUI, followed by 2D-Classification to select templates, then Relion auto-picking was used.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 208024 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingB value: 45.1 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: 0.92

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