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- EMDB-5678: Validated Near-Atomic Resolution Structure of Bacteriophage Epsil... -

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Basic information

Entry
Database: EMDB / ID: EMD-5678
TitleValidated Near-Atomic Resolution Structure of Bacteriophage Epsilon15 Derived from Cryo-EM and Modeling
Map dataReconstruction of infectious Epsilon15 bacteriophage.
Sample
  • Sample: Bacteriophage epsilon15
  • Virus: Salmonella phage epsilon15 (virus)
KeywordsCryo-EM / modeling / bacteriophage / validation / capsid / resolution / epsilon15 / random model / truly independent refinement / gold standard
Function / homologyviral capsid, decoration / viral capsid / Major coat protein / Major capsid protein
Function and homology information
Biological speciesSalmonella phage epsilon15 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsBaker ML / Hryc CF / Zhang Q / Wu W / Jakana J / Haase-Pettingell C / Afonine PV / Adams PD / King JA / Jiang W / Chiu W
CitationJournal: 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
DepositionMay 30, 2013-
Header (metadata) releaseJul 3, 2013-
Map releaseJul 10, 2013-
UpdateAug 14, 2013-
Current statusAug 14, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5.2
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 5.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j40
  • Surface level: 5.2
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j40
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5678.png

Downloads & links

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Map

FileDownload / File: emd_5678.map.gz / Format: CCP4 / Size: 1.4 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of infectious Epsilon15 bacteriophage.
Voxel sizeX=Y=Z: 1.1942 Å
Density
Contour LevelBy AUTHOR: 5.2 / Movie #1: 5.2
Minimum - Maximum-15.006737709999999 - 24.632560730000002
Average (Standard dev.)0.0086009 (±1.42201555)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-360-360-360
Dimensions720720720
Spacing720720720
CellA=B=C: 859.82404 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.19421.19421.1942
M x/y/z720720720
origin x/y/z0.0000.0000.000
length x/y/z859.824859.824859.824
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-360-360-360
NC/NR/NS720720720
D min/max/mean-15.00724.6330.009

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Supplemental data

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Sample components

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Entire : Bacteriophage epsilon15

EntireName: Bacteriophage epsilon15
Components
  • Sample: Bacteriophage epsilon15
  • Virus: Salmonella phage epsilon15 (virus)

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Supramolecule #1000: Bacteriophage epsilon15

SupramoleculeName: Bacteriophage epsilon15 / type: sample / ID: 1000 / Details: As described in Jiang, 2008 (EMDB:5003) / Number unique components: 2
Molecular weightExperimental: 22 MDa

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Supramolecule #1: Salmonella phage epsilon15

SupramoleculeName: Salmonella phage epsilon15 / type: virus / ID: 1 / NCBI-ID: 215158 / Sci species name: Salmonella phage epsilon15 / Database: NCBI / Virus type: VIRION / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Salmonella (bacteria) / synonym: BACTERIA(EUBACTERIA)
Molecular weightExperimental: 22 MDa
Virus shellShell ID: 1 / Name: Gp7 / Diameter: 700 Å / T number (triangulation number): 7

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5 / Details: 50 mM Tris-HCl, pH 7.5, 25 mM NaCl, 5 mM MgCl2
GridDetails: Quantifoil R2/2 grid
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 80 K / Instrument: FEI VITROBOT MARK II / Method: Blot before plunging.

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Electron microscopy

MicroscopeJEOL 3200FSC
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 53361 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 4.1 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 50000
Specialist opticsEnergy filter - Name: in-column filter / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 25.0 eV
Sample stageSpecimen holder model: JEOL 3200FSC CRYOHOLDER
TemperatureAverage: 81 K
DateJan 3, 2007
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.35 µm / Number real images: 1309 / Average electron dose: 17 e/Å2
Details: Digitized using Nikon Super CoolScan 9000 ED at 6.35 um/pixel
Bits/pixel: 12

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Image processing

CTF correctionDetails: per particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: OTHER / Software - Name: jspr, EMAN2, EMAN
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 4.5 Angstrom using the 0.143 threshold for the combined dataset.
Number images used: 14000
DetailsIndividual particles (720x720 pixels) were first automatically selected using the ethan method followed by manual screening using 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 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 6A. The final map of the entire dataset was then built from ~14000 particles by combining these two subsets of particles.

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