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- EMDB-10593: Cryo-EM structure of Pf4 bacteriophage coat protein with single-s... -

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Basic information

Entry
Database: EMDB / ID: EMD-10593
TitleCryo-EM structure of Pf4 bacteriophage coat protein with single-stranded DNA
Map data
Sample
  • Virus: Pseudomonas virus Pf1
    • Complex: Pseudomonas virus Pf1
      • Protein or peptide: Coat protein B of bacteriophage Pf1
    • Complex: DNA
      • DNA: DNA (5'-D(P*AP*AP*AP*AP*AP*A)-3')
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 / Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria) / Pseudomonas aeruginosa PAO1 (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsTarafder AK / von Kugelgen A / Bharat TAM
Funding support United Kingdom, 1 items
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, 2020-
Header (metadata) releaseFeb 26, 2020-
Map releaseFeb 26, 2020-
UpdateMar 11, 2020-
Current statusMar 11, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6tup
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6tup
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10593.png

Downloads & links

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Map

FileDownload / File: emd_10593.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.38 Å
Density
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.03785945 - 0.10565073
Average (Standard dev.)-0.000012020017 (±0.0022136576)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 552.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.381.381.38
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z552.000552.000552.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0380.106-0.000

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Supplemental data

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Sample components

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Entire : Pseudomonas virus Pf1

EntireName: Pseudomonas virus Pf1
Components
  • Virus: Pseudomonas virus Pf1
    • Complex: Pseudomonas virus Pf1
      • Protein or peptide: Coat protein B of bacteriophage Pf1
    • Complex: DNA
      • DNA: DNA (5'-D(P*AP*AP*AP*AP*AP*A)-3')

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Supramolecule #1: Pseudomonas virus Pf1

SupramoleculeName: Pseudomonas virus Pf1 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria)
Virus shellShell ID: 1 / Name: Coat protein B (CoaB) / Diameter: 62.0 Å

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Supramolecule #2: Pseudomonas virus Pf1

SupramoleculeName: Pseudomonas virus Pf1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Pseudomonas virus Pf1

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria)

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Macromolecule #1: Coat protein B of bacteriophage Pf1

MacromoleculeName: Coat protein B of bacteriophage Pf1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas virus Pf1
Molecular weightTheoretical: 4.612393 KDa
SequenceString:
GVIDTSAVES AITDGQGDMK AIGGYIVGAL VILAVAGLIY SMLRKA

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Macromolecule #2: DNA (5'-D(P*AP*AP*AP*AP*AP*A)-3')

MacromoleculeName: DNA (5'-D(P*AP*AP*AP*AP*AP*A)-3') / type: dna / ID: 2 / Details: Model of pf4 single-stranded DNA genome / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Molecular weightTheoretical: 1.834283 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DA)(DA)

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration5 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
8.0 g/lNaClSodium chloridesodium chloride
0.2 g/lKClpotassium chloride
1.15 g/lNa2HPO4sodium phosphate dibasic
0.2 g/lKH2PO4potassium phosphate monobasic

Details: 1x Phosphate buffered saline
GridModel: Quantifoil R2/2 / Material: COPPER/RHODIUM / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.03 kPa
Details: 20 second glow discharge at 15 mA in a LeicaEM ACE200
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV
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..
DetailsFilamentous phage purified from source

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: -3.0 µm / Calibrated defocus min: -1.0 µm / Calibrated magnification: 105000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -3.0 µm / Nominal defocus min: -1.0 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 80.0 K / Max: 80.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 2 / Number real images: 4110 / Average exposure time: 10.0 sec. / Average electron dose: 43.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 351381 / Software - Name: RELION (ver. 2.0)
CTF correctionSoftware - Name: RELION (ver. 2.0)
Details: Wiener filter implemented in the RELION refinement algorithm
Startup modelType of model: NONE / Details: Fourier-Bessel indexing
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 2.0)
Final reconstructionNumber classes used: 1
Applied symmetry - Helical parameters - Δz: 3.14 Å
Applied symmetry - Helical parameters - Δ&Phi: 65.9 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 185002

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Atomic model buiding 1

DetailsThe helical filament symmetry is only applied to the Pf4 coat protein and not the DNA. The reason for this is that the DNA bases are not resolved clearly due to averaging along the ssDNA genome of the phage, meaning that refinement of the protein into the map is of a higher quality than the ssDNA, where a poly-adenine model has been built.
RefinementSpace: REAL / Protocol: BACKBONE TRACE / Overall B value: 93.74 / Target criteria: Correlation coefficient
Output model

PDB-6tup:
Cryo-EM structure of Pf4 bacteriophage coat protein with single-stranded DNA

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