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- EMDB-6555: 2.9 Angstrom Resolution Cryo-EM 3-D Reconstruction of Close-packe... -

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Entry
Database: EMDB / ID: 6555
Title2.9 Angstrom Resolution Cryo-EM 3-D Reconstruction of Close-packed PCV2 Virus-like Particles
Map dataReconstruction of porcine circovirus 2 (PCV2) virus-like particles
SamplePorcine circovirus PCV2 virus-like particles:
virus
Keywordsde novo initial model / consensus criterion / gold-standard FSC / true FSC / cross-validation
Function / homologyCircovirus capsid protein / Circovirus capsid superfamily / Circovirus capsid protein / viral capsid assembly / host cell nucleus / Capsid protein / Capsid protein
Function and homology information
SourcePorcine circovirus 2 (PCV2)
Methodsingle particle reconstruction / cryo EM / 2.9 Å resolution
AuthorsLiu Z / Guo F / Wang F / Li TC / Jiang W
CitationJournal: Structure / Year: 2016
Title: 2.9 Å Resolution Cryo-EM 3D Reconstruction of Close-Packed Virus Particles.
Authors: Zheng Liu / Fei Guo / Feng Wang / Tian-Cheng Li / Wen Jiang
Abstract: Single-particle cryoelectron microscopy typically discards close-packed particle images as unusable data. Here, we report an image processing strategy and case study of obtaining near-atomic ...Single-particle cryoelectron microscopy typically discards close-packed particle images as unusable data. Here, we report an image processing strategy and case study of obtaining near-atomic resolution 3D reconstructions from close-packed particles. Multiple independent de novo initial models were constructed to determine and cross-validate the particle parameters. The particles with consistent views were further refined including not only Euler angles and center positions but also defocus, astigmatism, beam tilt, and overall and anisotropic magnification. We demonstrated this strategy with a 2.9 Å resolution reconstruction of a 1.67 MDa virus-like particle of a circovirus, PCV2, recorded on 86 photographic films. The map resolution was further validated with a phase-randomization test and local resolution assessment, and the atomic model was validated with MolProbity and EMRinger. Close-packed virus particles were thus shown not only to be useful for high-resolution 3D reconstructions but also to allow data collection at significantly improved throughput for near-atomic resolution reconstructions.
Validation ReportPDB-ID: 3jci

SummaryFull reportAbout validation report
DateDeposition: Dec 7, 2015 / Header (metadata) release: Feb 3, 2016 / Map release: Feb 3, 2016 / Last update: Mar 30, 2016

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 10
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 10
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-3jci
  • Surface level: 10
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-3jci
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_6555.map.gz (map file in CCP4 format, 128001 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
320 pix
1.08 Å/pix.
= 345.6 Å
320 pix
1.08 Å/pix.
= 345.6 Å
320 pix
1.08 Å/pix.
= 345.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density
Contour Level:10 (by author), 10 (movie #1):
Minimum - Maximum-33.87968063 - 57.06698990
Average (Standard dev.)0.08360518 (2.59643769)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions320320320
Origin-160-160-160
Limit159159159
Spacing320320320
CellA=B=C: 345.6 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z345.600345.600345.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-34-31-64
NX/NY/NZ6963129
MAP C/R/S123
start NC/NR/NS-160-160-160
NC/NR/NS320320320
D min/max/mean-33.88057.0670.084

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Supplemental data

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Sample components

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Entire Porcine circovirus PCV2 virus-like particles

EntireName: Porcine circovirus PCV2 virus-like particles / Number of components: 1
MassTheoretical: 1.67 MDa / Experimental: 1.67 MDa

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Component #1: virus, Porcine circovirus 2

VirusName: Porcine circovirus 2 / a.k.a: PCV2 / Class: VIRUS-LIKE PARTICLE / Empty: Yes / Enveloped: No / Isolate: STRAIN
MassTheoretical: 1.67 MDa / Experimental: 1.67 MDa
SpeciesSpecies: Porcine circovirus 2 (PCV2) / Strain: Yamagata
Source (engineered)Expression System: Trichoplusia ni (cabbage looper) / Vector: AcPCV2-ORF2 / Cell of expression system: Tn5
Source (natural)Host Species: Sus scrofa (pig) / Host category: VERTEBRATES

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 3 mg/ml / Buffer solution: 1x PBS / pH: 7.4
Support film400-mesh holey carbon grids (1.2/1.3 C-flat, Protochips)
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Temperature: 85 K / Humidity: 90 % / Method: Blot for 5 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Feb 22, 2011
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 25 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 59000 X (nominal), 587963 X (calibrated)
Astigmatism: Objective lens astigmatism was corrected at 250,000 magnification using a quadrupole stigmator.
Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 200 - 2500 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: 90 K ( 80 - 100 K)
CameraDetector: KODAK SO-163 FILM

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Image acquisition

Image acquisitionNumber of digital images: 141 / Scanner: NIKON SUPER COOLSCAN 9000 / Sampling size: 6.35 microns / Bit depth: 16 / OD range: 1

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: I (icosahedral) / Number of projections: 50352
Details: The particles were selected using the e2boxer.py program in EMAN2. CTF parameters were determined using fitctf2.py in JSPR.
3D reconstructionAlgorithm: Projection matching / Software: JSPR, EMAN2 / CTF correction: Each particle
Details: For 3D reconstruction, whole datasets were divided into even and odd halves and the initial de novo models and subsequent iterative refinements were all independently performed for each half dataset. Particles were selected from scanned micrograph images using e2boxer.py in EMAN2. The TEM instrument contrast transfer function parameters were determined automatically using fitctf2.py in JSPR and were then visually validated using the EMAN ctfit program. The datasets were then divided into two subsets (even and odd) and processed completely independently, including both de novo initial models and refinements. The images were first binned 4x to obtain initial models and particle parameters assuming icosahedral symmetry. De novo initial models were built using the random model approach. Random subsets of particles were assigned random initial orientations and iteratively refined until convergence. Multi-model competitive refinements were used to choose the winning model (with most assigned particles) as corrective initial models for subsequent refinement. Particles with inconsistent/unstable view parameters in the initial refinements were excluded in further image processing. The orientation and center parameters were then transferred to the un-binned images for high-resolution refinements which included Simplex method-based orientation/center optimization and grid search-based refinement of defocus, astigmatism, beam tilt, and overall and anisotropic magnification of the images. All image refinement and reconstructions were performed with JSPR software that was built on EMAN2 and EMAN library functions and programs.
Resolution: 2.9 Å / Resolution method: FSC 0.143, gold-standard

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