|Entry||Database: EMDB / ID: 6555|
|Title||2.9 Angstrom Resolution Cryo-EM 3-D Reconstruction of Close-packed PCV2 Virus-like Particles|
|Map data||Reconstruction of porcine circovirus 2 (PCV2) virus-like particles|
|Sample||Porcine circovirus PCV2 virus-like particles:|
|Keywords||de novo initial model / consensus criterion / gold-standard FSC / true FSC / cross-validation|
|Function / homology||Circovirus capsid protein / Circovirus capsid superfamily / Circovirus capsid protein / viral capsid assembly / host cell nucleus / Capsid protein / Capsid protein|
Function and homology information
|Source||Porcine circovirus 2 (PCV2)|
|Method||single particle reconstruction / cryo EM / 2.9 Å resolution|
|Authors||Liu Z / Guo F / Wang F / Li TC / Jiang W|
|Citation||Journal: 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 Report||PDB-ID: 3jci|
SummaryFull reportAbout validation report
|Date||Deposition: Dec 7, 2015 / Header (metadata) release: Feb 3, 2016 / Map release: Feb 3, 2016 / Last update: Mar 30, 2016|
|Structure viewer||EM map: |
Downloads & links
|File||emd_6555.map.gz (map file in CCP4 format, 128001 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.08 Å|
CCP4 map header:
-Entire Porcine circovirus PCV2 virus-like particles
|Entire||Name: Porcine circovirus PCV2 virus-like particles / Number of components: 1|
|Mass||Theoretical: 1.67 MDa / Experimental: 1.67 MDa|
-Component #1: virus, Porcine circovirus 2
|Virus||Name: Porcine circovirus 2 / a.k.a: PCV2 / Class: VIRUS-LIKE PARTICLE / Empty: Yes / Enveloped: No / Isolate: STRAIN|
|Mass||Theoretical: 1.67 MDa / Experimental: 1.67 MDa|
|Species||Species: 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|
|Specimen||Specimen state: particle / Method: cryo EM|
|Sample solution||Specimen conc.: 3 mg/ml / Buffer solution: 1x PBS / pH: 7.4|
|Support film||400-mesh holey carbon grids (1.2/1.3 C-flat, Protochips)|
|Vitrification||Instrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Temperature: 85 K / Humidity: 90 % / Method: Blot for 5 seconds before plunging.|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS / Date: Feb 22, 2011|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 25 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 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 Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: 90 K ( 80 - 100 K)|
|Camera||Detector: KODAK SO-163 FILM|
|Image acquisition||Number of digital images: 141 / Scanner: NIKON SUPER COOLSCAN 9000 / Sampling size: 6.35 microns / Bit depth: 16 / OD range: 1|
|Processing||Method: 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 reconstruction||Algorithm: 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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