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- PDB-3jci: 2.9 Angstrom Resolution Cryo-EM 3-D Reconstruction of Close-packe... -

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Basic information

Entry
Database: PDB / ID: 3jci
Title2.9 Angstrom Resolution Cryo-EM 3-D Reconstruction of Close-packed PCV2 Virus-like Particles
ComponentsCapsid proteinCapsid
KeywordsVIRUS LIKE PARTICLE / de 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 protein / Circovirus capsid superfamily / Circovirus capsid protein / Jelly Rolls / Sandwich / Mainly Beta
Similarity search - Domain/homology
Capsid protein / Capsid protein
Similarity search - Component
Biological speciesPorcine circovirus-2
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsLiu, Z. / Guo, F. / Wang, F. / Li, T.C. / 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 13, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 3, 2016Provider: repository / Type: Initial release
Revision 1.1Feb 17, 2016Group: Database references
Revision 1.2Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id / _em_software.name
Revision 1.3Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.4Feb 21, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

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  • Biological unit as complete icosahedral assembly
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  • Biological unit as icosahedral pentamer
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  • Biological unit as icosahedral 23 hexamer
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-6555
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Capsid protein


Theoretical massNumber of molelcules
Total (without water)22,0001
Polymers22,0001
Non-polymers00
Water0
1
A: Capsid protein
x 60


Theoretical massNumber of molelcules
Total (without water)1,319,98560
Polymers1,319,98560
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
2


  • Idetical with deposited unit
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
A: Capsid protein
x 5


  • icosahedral pentamer
  • 110 kDa, 5 polymers
Theoretical massNumber of molelcules
Total (without water)109,9995
Polymers109,9995
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: Capsid protein
x 6


  • icosahedral 23 hexamer
  • 132 kDa, 6 polymers
Theoretical massNumber of molelcules
Total (without water)131,9996
Polymers131,9996
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein Capsid protein / Capsid


Mass: 21999.758 Da / Num. of mol.: 1 / Fragment: UNP residues 42-231 / Source method: isolated from a natural source / Source: (natural) Porcine circovirus-2 / References: UniProt: B8Y5Y9, UniProt: Q8B980*PLUS

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeParent-ID
1Porcine circovirus PCV2 virus-like particlesVIRUS0
2Porcine circovirus 2 (PCV2)VIRUS1
Molecular weightValue: 1.67 MDa / Experimental value: NO
Details of virusEmpty: YES / Enveloped: NO / Host category: VERTEBRATES / Isolate: STRAIN / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Sus scrofa
Buffer solutionName: PBS / pH: 7.4 / Details: PBS
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 400-mesh holey carbon grids (1.2/1.3 C-flat, Protochips)
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Temp: 85 K / Humidity: 90 %
Details: Blot for 5 seconds before plunging into liquid ethane (GATAN CRYOPLUNGE 3).
Method: Blot for 5 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Feb 22, 2011
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Calibrated magnification: 587963 X / Nominal defocus max: 2500 nm / Nominal defocus min: 200 nm / Cs: 2.7 mm
Astigmatism: Objective lens astigmatism was corrected at 250,000 magnification using quadrupole stigmator.
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: 90 K / Temperature (max): 100 K / Temperature (min): 80 K
Image recordingElectron dose: 25 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 141

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Processing

EM software
IDNameVersionCategory
1EMAN23D reconstruction
2jspr3D reconstruction
CTF correctionDetails: Each particle
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionMethod: Projection matching / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50352 / Nominal pixel size: 1.1 Å / Actual pixel size: 1.08 Å
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. (Single particle details: The particles were selected using the e2boxer.py program in EMAN2. CTF parameters were determined using fitctf2.py in JSPR.) (Single particle--Applied symmetry: I)
Symmetry type: POINT
Displacement parametersBiso max: 77.94 Å2 / Biso mean: 23.672 Å2 / Biso min: 10.28 Å2
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms1557 0 0 0 1557
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.011604
ELECTRON MICROSCOPYf_angle_d0.8172187
ELECTRON MICROSCOPYf_chiral_restr0.054233
ELECTRON MICROSCOPYf_plane_restr0.006285
ELECTRON MICROSCOPYf_dihedral_angle_d17.1511291

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