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- PDB-7ofh: CryoEM structure of the outer membrane secretin pore pIV from the... -

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Basic information

Entry
Database: PDB / ID: 7ofh
TitleCryoEM structure of the outer membrane secretin pore pIV from the f1 filamentous bacteriophage.
ComponentsVirion export protein
KeywordsVIRAL PROTEIN / Secretin Outer membrane Virion export
Function / homology
Function and homology information


viral extrusion / protein secretion / host cell membrane / membrane => GO:0016020
Similarity search - Function
GspD/PilQ family / Bacterial type II secretion system protein D signature. / Type II secretion system protein GspD, conserved site / NolW-like superfamily / Type II/III secretion system / Bacterial type II and III secretion system protein
Similarity search - Domain/homology
Virion export protein
Similarity search - Component
Biological speciesEnterobacteria phage f1 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsConners, R. / Gold, V.A.M.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust210363/Z/18/Z United Kingdom
CitationJournal: Nat Commun / Year: 2021
Title: CryoEM structure of the outer membrane secretin channel pIV from the f1 filamentous bacteriophage.
Authors: Rebecca Conners / Mathew McLaren / Urszula Łapińska / Kelly Sanders / M Rhia L Stone / Mark A T Blaskovich / Stefano Pagliara / Bertram Daum / Jasna Rakonjac / Vicki A M Gold /
Abstract: The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has ...The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 Å resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.
History
DepositionMay 5, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 3, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 17, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_proc
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: Virion export protein
B: Virion export protein
C: Virion export protein
D: Virion export protein
E: Virion export protein
F: Virion export protein
G: Virion export protein
H: Virion export protein
I: Virion export protein
J: Virion export protein
K: Virion export protein
L: Virion export protein
M: Virion export protein
N: Virion export protein
O: Virion export protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)688,31245
Polymers669,86615
Non-polymers18,44630
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy, 15 fold symmetry was observed in the 2D classes obtained from Relion
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area93790 Å2
ΔGint-448 kcal/mol
Surface area154320 Å2
MethodPISA

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Components

#1: Protein
Virion export protein / Gene 4 protein / G4P


Mass: 44657.707 Da / Num. of mol.: 15 / Mutation: S318I
Source method: isolated from a genetically manipulated source
Details: S9P, D49N, I66N are naturally occurring polymorphisms.
Source: (gene. exp.) Enterobacteria phage f1 (virus) / Gene: IV / Variant: S9P, D49N, I66N / Plasmid: pPMR132 / Production host: Escherichia coli (E. coli) / Variant (production host): TG1 / References: UniProt: P03666
#2: Chemical...
ChemComp-CPS / 3-[(3-CHOLAMIDOPROPYL)DIMETHYLAMMONIO]-1-PROPANESULFONATE / CHAPS / CHAPS detergent


Mass: 614.877 Da / Num. of mol.: 30 / Source method: obtained synthetically / Formula: C32H58N2O7S / Comment: detergent*YM
Has ligand of interestN
Nonpolymer detailsResidue 501 is full-length CHAPS, and residue 502 has been modeled as the CHAPS aromatic rings ...Residue 501 is full-length CHAPS, and residue 502 has been modeled as the CHAPS aromatic rings only, due to there being no clear density for the rest of the CHAPS molecule.

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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: Virion export protein pIV / Type: ORGANELLE OR CELLULAR COMPONENT / Details: From Inoviridae sp. f1 / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.6697 MDa / Experimental value: NO
Source (natural)Organism: Inoviridae sp. (virus)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: TG1 / Plasmid: pPMR132
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
11 % (w/v)CHAPS (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate)C32H58N2O7S1
225 mMsodium hepesC8H17N2NaO4S1
3500 mMsodium chlorideNaClSodium chloride1
40.5 mMEDTAEthylenediaminetetraacetic acidC10H16N2O81
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid mesh size: 300 divisions/in. / Grid type: PELCO Ultrathin Carbon with Lacey Carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot force 0, blot time 4sec

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X
Image recordingAverage exposure time: 3.52 sec. / Electron dose: 42.059 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
1Warpparticle selectionParticle selection
2EPUimage acquisitionData collection
4WarpCTF correction
7Cootmodel fitting
9RELIONinitial Euler assignment
10RELION3.1betafinal Euler assignment
11RELIONclassification
12RELION3.1beta3D reconstruction
13REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C15 (15 fold cyclic)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 111679 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 5W68

5w68

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