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Yorodumi- PDB-3j40: Validated Near-Atomic Resolution Structure of Bacteriophage Epsil... -
+Open data
-Basic information
Entry | Database: PDB / ID: 3j40 | |||||||||
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Title | Validated Near-Atomic Resolution Structure of Bacteriophage Epsilon15 Derived from Cryo-EM and Modeling | |||||||||
Components |
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Keywords | VIRUS / capsid / accessory protein | |||||||||
Function / homology | : / Major coat protein-like / : / Major capsid protein GP7 / viral capsid, decoration / viral capsid / Major coat protein / Major capsid protein Function and homology information | |||||||||
Biological species | Salmonella phage epsilon15 (virus) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å | |||||||||
Authors | Baker, M.L. / Hryc, C.F. / Zhang, Q. / Wu, W. / Jakana, J. / Haase-Pettingell, C. / Afonine, P.V. / Adams, P.D. / King, J.A. / Jiang, W. / Chiu, W. | |||||||||
Citation | Journal: Proc Natl Acad Sci U S A / Year: 2013 Title: Validated near-atomic resolution structure of bacteriophage epsilon15 derived from cryo-EM and modeling. Authors: Matthew L Baker / Corey F Hryc / Qinfen Zhang / Weimin Wu / Joanita Jakana / Cameron Haase-Pettingell / Pavel V Afonine / Paul D Adams / Jonathan A King / Wen Jiang / Wah Chiu / Abstract: High-resolution structures of viruses have made important contributions to modern structural biology. Bacteriophages, the most diverse and abundant organisms on earth, replicate and infect all ...High-resolution structures of viruses have made important contributions to modern structural biology. Bacteriophages, the most diverse and abundant organisms on earth, replicate and infect all bacteria and archaea, making them excellent potential alternatives to antibiotics and therapies for multidrug-resistant bacteria. Here, we improved upon our previous electron cryomicroscopy structure of Salmonella bacteriophage epsilon15, achieving a resolution sufficient to determine the tertiary structures of both gp7 and gp10 protein subunits that form the T = 7 icosahedral lattice. This study utilizes recently established best practice for near-atomic to high-resolution (3-5 Å) electron cryomicroscopy data evaluation. The resolution and reliability of the density map were cross-validated by multiple reconstructions from truly independent data sets, whereas the models of the individual protein subunits were validated adopting the best practices from X-ray crystallography. Some sidechain densities are clearly resolved and show the subunit-subunit interactions within and across the capsomeres that are required to stabilize the virus. The presence of the canonical phage and jellyroll viral protein folds, gp7 and gp10, respectively, in the same virus suggests that epsilon15 may have emerged more recently relative to other bacteriophages. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 3j40.cif.gz | 605.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb3j40.ent.gz | 498.2 KB | Display | PDB format |
PDBx/mmJSON format | 3j40.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 3j40_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 3j40_full_validation.pdf.gz | 1.3 MB | Display | |
Data in XML | 3j40_validation.xml.gz | 107.3 KB | Display | |
Data in CIF | 3j40_validation.cif.gz | 157.2 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/j4/3j40 ftp://data.pdbj.org/pub/pdb/validation_reports/j4/3j40 | HTTPS FTP |
-Related structure data
Related structure data | 5003M 5678MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
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Symmetry | Point symmetry: (Schoenflies symbol: I (icosahedral)) |
-Components
#1: Protein | Mass: 12181.435 Da / Num. of mol.: 7 / Source method: isolated from a natural source / Source: (natural) Salmonella phage epsilon15 (virus) / References: UniProt: Q858G5 #2: Protein | Mass: 36856.453 Da / Num. of mol.: 7 / Source method: isolated from a natural source / Source: (natural) Salmonella phage epsilon15 (virus) / References: UniProt: Q858G8 |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Bacteriophage epsilon15 / Type: VIRUS |
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Molecular weight | Value: 22 MDa / Experimental value: YES |
Details of virus | Empty: NO / Enveloped: NO / Host category: BACTERIA(EUBACTERIA) / Isolate: SPECIES / Type: VIRION |
Natural host | Organism: Salmonella |
Buffer solution | Name: 50 mM Tris, pH 7.5, 25 mM NaCl, 10 mM MgCl2 / pH: 7.5 / Details: 50 mM Tris, pH 7.5, 25 mM NaCl, 10 mM MgCl2 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Details: Quantifoil R2/2 grid |
Vitrification | Instrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Temp: 80 K / Humidity: 90 % Details: Blot before plunging into liquid ethane (FEI VITROBOT MARK II). Method: Blot before plunging. |
-Electron microscopy imaging
Microscopy | Model: JEOL 3200FSC / Date: Jan 3, 2007 |
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Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 50000 X / Calibrated magnification: 53361 X / Nominal defocus max: 2700 nm / Nominal defocus min: 400 nm / Cs: 4.1 mm / Camera length: 0 mm |
Specimen holder | Specimen holder model: JEOL 3200FSC CRYOHOLDER / Temperature: 81 K / Tilt angle max: 0 ° / Tilt angle min: 0 ° |
Image recording | Electron dose: 17 e/Å2 / Film or detector model: KODAK SO-163 FILM |
EM imaging optics | Energyfilter name: in-column filter / Energyfilter upper: 25 eV / Energyfilter lower: 0 eV |
Image scans | Num. digital images: 1309 |
Radiation | Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray |
Radiation wavelength | Relative weight: 1 |
-Processing
EM software |
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CTF correction | Details: per particle | ||||||||||||||||
Symmetry | Point symmetry: I (icosahedral) | ||||||||||||||||
3D reconstruction | Method: icosahedral / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 14000 / Nominal pixel size: 1.194 Å / Actual pixel size: 1.1942 Å Details: The gold standard definition for the resolution estimate was adopted whereby the particle images were split into two subsets at the onset of image processing and the datasets were ...Details: The gold standard definition for the resolution estimate was adopted whereby the particle images were split into two subsets at the onset of image processing and the datasets were individually reconstructed and then combined after determination of the resolution estimate. Independent initial models were built de novo and used for the subsequent particle refinements in each of the two subsets of particle images. The Fourier Shell Correlation (FSC) between the two independently determined reconstructions was computed and indicated a resolution of 4.5 Angstrom using the 0.143 threshold for the combined dataset. (Single particle details: Individual particles (720x720 pixels) were first automatically selected using the ethan method followed by manual screening using the EMAN boxer program. A total of 54161 particles were selected for initial processing. The selected particles within a micrograph were incoherently averaged to generate 2D power spectra for contrast transfer function (CTF) parameter determination. CTF parameters were first automatically estimated and then visually verified using the EMAN1 ctfit program. Defocus values range from 0.5 to 2.5 um. The data set was divided into two data subsets for the following reconstruction steps. The particle images were first binned 4x for initial model building and initial determination of orientation and center parameters. The initial model was built de novo by iterative refinement of a subset of 300 particles randomly selected from the half data set with randomly assigned initial orientations. The initial orientations of all particles in each of the half data sets were determined using the EMAN1 projection matching program classesbymra with an angular projection step size of 3 degrees. The orientations were then refined to higher accuracy using the program jalign, which is based on simplex optimization of matching between the particle image and model projections. The particle orientation parameters were then transferred to particles binned at 2x and ultimately to particles without binning for further refinements. In the last stage of refinement, magnification, astigmatism, and defocus parameters were also included. 3D maps with icosahedral symmetry enforcement were reconstructed using a newly developed program, j3dr, using the EMAN2 library and parallelized with message passing interface (MPI) to speed up the reconstruction process. These steps were iterated until the refinement converged. The map for each data subset was reconstructed from ~7000 particles by removing particles with poor alignment scores and unstable alignment parameters. The resolution of the map was evaluated using the Fourier Shell Correlation (FSC). Only the icosahedral shell region was included in this FSC analysis by masking out the external background noises and the internal DNA densities using soft masks with a half width of 6 Angstrom. The final map of the entire dataset was then built from ~14000 particles by combining these two subsets of particles.) (Single particle--Applied symmetry: I) Refinement type: HALF-MAPS REFINED INDEPENDENTLY / Symmetry type: POINT | ||||||||||||||||
Refinement step | Cycle: LAST
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