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- PDB-6tup: Cryo-EM structure of Pf4 bacteriophage coat protein with single-s... -
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Open data
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Basic information
Entry | Database: PDB / ID: 6tup | ||||||
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Title | Cryo-EM structure of Pf4 bacteriophage coat protein with single-stranded DNA | ||||||
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![]() | VIRUS / Bacteriophage / helical / filamentous | ||||||
Function / homology | Inovirus Coat protein B / Capsid protein G8P / helical viral capsid / host cell membrane / membrane / DNA / Capsid protein G8P / Coat protein B of bacteriophage Pf1![]() | ||||||
Biological species | ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.2 Å | ||||||
![]() | Tarafder, A.K. / von Kugelgen, A. / Bharat, T.A.M. | ||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 25 KB | Display | ![]() |
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PDB format | ![]() | 14.2 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 10593MC ![]() 6tuqC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein/peptide | Mass: 4612.393 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() |
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#2: DNA chain | Mass: 1834.283 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Model of pf4 single-stranded DNA genome / Source: (natural) ![]() |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: FILAMENT / 3D reconstruction method: helical reconstruction |
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Sample preparation
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Molecular weight | Experimental value: NO | |||||||||||||||||||||||||
Source (natural) |
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Details of virus | Empty: NO / Enveloped: NO / Isolate: STRAIN / Type: VIRION | |||||||||||||||||||||||||
Natural host | Organism: Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) | |||||||||||||||||||||||||
Virus shell | Name: Coat protein B (CoaB) / Diameter: 62 nm | |||||||||||||||||||||||||
Buffer solution | pH: 7.4 / Details: 1x Phosphate buffered saline | |||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Filamentous phage purified from source | |||||||||||||||||||||||||
Specimen support | Details: 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 | |||||||||||||||||||||||||
Vitrification | Instrument: 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 |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: 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 holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 K / Temperature (min): 80 K |
Image recording | Average 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 optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
Image scans | Movie frames/image: 40 / Used frames/image: 1-40 |
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Processing
EM software |
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CTF correction | Details: Wiener filter implemented in the RELION refinement algorithm Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Helical symmerty | Angular rotation/subunit: 65.9 ° / Axial rise/subunit: 3.14 Å / Axial symmetry: C1 | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 351381 | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 185002 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: HELICAL | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 93.74 / Protocol: BACKBONE TRACE / Space: REAL / Target criteria: Correlation coefficient Details: The 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 ...Details: The 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. |