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- PDB-6tuq: Cryo-EM structure of Pf4 bacteriophage coat protein without ssDNA -

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Basic information

Entry
Database: PDB / ID: 6tuq
TitleCryo-EM structure of Pf4 bacteriophage coat protein without ssDNA
ComponentsCoat protein B of bacteriophage Pf1
KeywordsVIRUS / Bacteriophage / helical / filamentous
Function / homologyInovirus Coat protein B / Capsid protein G8P / helical viral capsid / host cell membrane / membrane / Capsid protein G8P / Coat protein B of bacteriophage Pf1
Function and homology information
Biological speciesPseudomonas virus Pf1
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsTarafder, A.K. / von Kugelgen, A. / Bharat, T.A.M.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust202231/Z/16/Z United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Phage liquid crystalline droplets form occlusive sheaths that encapsulate and protect infectious rod-shaped bacteria.
Authors: Abul K Tarafder / Andriko von Kügelgen / Adam J Mellul / Ulrike Schulze / Dirk G A L Aarts / Tanmay A M Bharat /
Abstract: The opportunistic pathogen is a major cause of antibiotic-tolerant infections in humans. evades antibiotics in bacterial biofilms by up-regulating expression of a symbiotic filamentous inoviral ...The opportunistic pathogen is a major cause of antibiotic-tolerant infections in humans. evades antibiotics in bacterial biofilms by up-regulating expression of a symbiotic filamentous inoviral prophage, Pf4. We investigated the mechanism of phage-mediated antibiotic tolerance using biochemical reconstitution combined with structural biology and high-resolution cellular imaging. We resolved electron cryomicroscopy atomic structures of Pf4 with and without its linear single-stranded DNA genome, and studied Pf4 assembly into liquid crystalline droplets using optical microscopy and electron cryotomography. By biochemically replicating conditions necessary for antibiotic protection, we found that phage liquid crystalline droplets form phase-separated occlusive compartments around rod-shaped bacteria leading to increased bacterial survival. Encapsulation by these compartments was observed even when inanimate colloidal rods were used to mimic rod-shaped bacteria, suggesting that shape and size complementarity profoundly influences the process. Filamentous inoviruses are pervasive across prokaryotes, and in particular, several Gram-negative bacterial pathogens including , and harbor these prophages. We propose that biophysical occlusion mediated by secreted filamentous molecules such as Pf4 may be a general strategy of bacterial survival in harsh environments.
History
DepositionJan 8, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 26, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 4, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Mar 11, 2020Group: Data collection / Database references / Category: citation / em_imaging_optics
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_imaging_optics.phase_plate
Revision 1.3May 22, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation

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Structure visualization

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Assembly

Deposited unit
A: Coat protein B of bacteriophage Pf1


Theoretical massNumber of molelcules
Total (without water)4,6121
Polymers4,6121
Non-polymers00
Water00
1
A: Coat protein B of bacteriophage Pf1
x 50


Theoretical massNumber of molelcules
Total (without water)230,62050
Polymers230,62050
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation49

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Components

#1: Protein/peptide Coat protein B of bacteriophage Pf1 / Phage coat protein B


Mass: 4612.393 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Pseudomonas virus Pf1 / References: UniProt: Q9I5K5, UniProt: P03621*PLUS

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Pseudomonas virus Pf1 / Type: VIRUS / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Pseudomonas virus Pf1
Details of virusEmpty: YES / Enveloped: NO / Isolate: STRAIN / Type: VIRION
Natural hostOrganism: Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
Virus shellName: Coat protein B (CoaB) / Diameter: 62 nm
Buffer solutionpH: 7.4 / Details: 1x Phosphate buffered saline
Buffer component
IDConc.NameFormulaBuffer-ID
18 g/lsodium chlorideNaCl1
20.2 g/lpotassium chlorideKCl1
31.15 g/lsodium phosphate dibasicNa2HPO41
40.2 g/lpotassium phosphate monobasicKH2PO41
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 20 second glow discharge at 15 mA in a LeicaEM ACE200
Grid material: COPPER/RHODIUM / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K
Details: Samples for cryo-EM were prepared by pipetting 2.5 ul of the sample onto freshly glow-discharged Quantifoil grids (Cu/Rh R2/2, 200 mesh). Grids were blotted for 2.5 seconds with a blot force ...Details: Samples for cryo-EM were prepared by pipetting 2.5 ul of the sample onto freshly glow-discharged Quantifoil grids (Cu/Rh R2/2, 200 mesh). Grids were blotted for 2.5 seconds with a blot force of -15, 0.5 second drain and 0 second wait times.

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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 FIELD / Nominal magnification: 105000 X / Calibrated magnification: 105000 X / Nominal defocus max: -3000 nm / Nominal defocus min: -1000 nm / Calibrated defocus min: -1000 nm / Calibrated defocus max: -3000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 K / Temperature (min): 80 K
Image recordingAverage exposure time: 10 sec. / Electron dose: 43 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 4110
EM imaging opticsEnergyfilter name: GIF Bioquantum / Chromatic aberration corrector: none / Energyfilter slit width: 20 eV / Phase plate: OTHER / Spherical aberration corrector: none
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
1RELION2particle selection
2EPUimage acquisition
4RELION2CTF correction
7Cootmodel fitting
9RELION2initial Euler assignment
10RELION2final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIXmodel refinement
CTF correctionDetails: Wiener filter implemented in the RELION refinement algorithm
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 3.14 ° / Axial rise/subunit: 65.9 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 351381
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 95481 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: HELICAL
Atomic model buildingB value: 180.97 / Protocol: BACKBONE TRACE / Space: REAL / Target criteria: Correlation coefficient

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