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

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Basic information

Entry
Database: EMDB / ID: EMD-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
Sample
  • Sample: Porcine circovirus PCV2 virus-like particles
  • Virus: Porcine circovirus 2
Keywordsde novo initial model / consensus criterion / gold-standard FSC / true FSC / cross-validation
Function / homology
Function and homology information


viral capsid assembly / T=1 icosahedral viral capsid / endocytosis involved in viral entry into host cell / viral penetration into host nucleus / host cell nucleus / virion attachment to host cell / nucleus
Similarity search - Function
Circovirus capsid protein / Circovirus capsid superfamily / Circovirus capsid protein
Similarity search - Domain/homology
Capsid protein / Capsid protein
Similarity search - Component
Biological speciesPorcine circovirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
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.
History
DepositionDec 7, 2015-
Header (metadata) releaseFeb 3, 2016-
Map releaseFeb 3, 2016-
UpdateMar 30, 2016-
Current statusMar 30, 2016Processing site: RCSB / Status: Released

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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 modelsPDB-3jci
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6555.png

Downloads & links

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Map

FileDownload / File: emd_6555.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of porcine circovirus 2 (PCV2) virus-like particles
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 10.0 / Movie #1: 10
Minimum - Maximum-33.879680630000003 - 57.066989900000003
Average (Standard dev.)0.08360518 (±2.59643769)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-160-160-160
Dimensions320320320
Spacing320320320
CellA=B=C: 345.6 Å
α=β=γ: 90.0 °

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
Components
  • Sample: Porcine circovirus PCV2 virus-like particles
  • Virus: Porcine circovirus 2

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Supramolecule #1000: Porcine circovirus PCV2 virus-like particles

SupramoleculeName: Porcine circovirus PCV2 virus-like particles / type: sample / ID: 1000 / Number unique components: 1
Molecular weightExperimental: 1.67 MDa / Theoretical: 1.67 MDa

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Supramolecule #1: Porcine circovirus 2

SupramoleculeName: Porcine circovirus 2 / type: virus / ID: 1 / Name.synonym: PCV2 / NCBI-ID: 85708 / Sci species name: Porcine circovirus 2 / Sci species strain: Yamagata / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: Yes / Syn species name: PCV2
Host (natural)Organism: Sus scrofa (pig) / synonym: VERTEBRATES
Host systemOrganism: Trichoplusia ni (cabbage looper) / Recombinant cell: Tn5 / Recombinant plasmid: AcPCV2-ORF2
Molecular weightExperimental: 1.67 MDa / Theoretical: 1.67 MDa
Virus shellShell ID: 1 / Diameter: 190 Å / T number (triangulation number): 1

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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

Concentration3 mg/mL
BufferpH: 7.4 / Details: 1x PBS
GridDetails: 400-mesh holey carbon grids (1.2/1.3 C-flat, Protochips)
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 85 K / Instrument: GATAN CRYOPLUNGE 3 / Method: Blot for 5 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.2 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureMin: 80 K / Max: 100 K / Average: 90 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 250,000 magnification using a quadrupole stigmator.
DateFeb 22, 2011
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: 141 / Average electron dose: 25 e/Å2 / Od range: 1 / Bits/pixel: 16
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: OTHER / Software - Name: JSPR, EMAN2
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 ...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.
Number images used: 50352
DetailsThe particles were selected using the e2boxer.py program in EMAN2. CTF parameters were determined using fitctf2.py in JSPR.

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