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- PDB-5fj5: Structure of the in vitro assembled bacteriophage phi6 polymerase... -

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Basic information

Entry
Database: PDB / ID: 5fj5
TitleStructure of the in vitro assembled bacteriophage phi6 polymerase complex
ComponentsMAJOR INNER PROTEIN P1
KeywordsVIRAL PROTEIN / POLYMERASE COMPLEX
Function / homologyT=2 icosahedral viral capsid / viral inner capsid / virion component => GO:0044423 / viral nucleocapsid / RNA binding / identical protein binding / Major inner protein P1
Function and homology information
Biological speciesPSEUDOMONAS PHAGE PHI6 (bacteriophage)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsIlca, S. / Kotecha, A. / Sun, X. / Poranen, M.P. / Stuart, D.I. / Huiskonen, J.T.
CitationJournal: Nat Commun / Year: 2015
Title: Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes.
Authors: Serban L Ilca / Abhay Kotecha / Xiaoyu Sun / Minna M Poranen / David I Stuart / Juha T Huiskonen /
Abstract: Electron cryomicroscopy can yield near-atomic resolution structures of highly ordered macromolecular complexes. Often however some subunits bind in a flexible manner, have different symmetry from the ...Electron cryomicroscopy can yield near-atomic resolution structures of highly ordered macromolecular complexes. Often however some subunits bind in a flexible manner, have different symmetry from the rest of the complex, or are present in sub-stoichiometric amounts, limiting the attainable resolution. Here we report a general method for the localized three-dimensional reconstruction of such subunits. After determining the particle orientations, local areas corresponding to the subunits can be extracted and treated as single particles. We demonstrate the method using three examples including a flexible assembly and complexes harbouring subunits with either partial occupancy or mismatched symmetry. Most notably, the method allows accurate fitting of the monomeric RNA-dependent RNA polymerase bound at the threefold axis of symmetry inside a viral capsid, revealing for the first time its exact orientation and interactions with the capsid proteins. Localized reconstruction is expected to provide novel biological insights in a range of challenging biological systems.
Validation Report
SummaryFull reportAbout validation report
History
DepositionOct 6, 2015Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 4, 2015Provider: repository / Type: Initial release
Revision 1.1Nov 18, 2015Group: Database references
Revision 1.2Aug 2, 2017Group: Data collection / Category: em_software
Item: _em_software.fitting_id / _em_software.image_processing_id / _em_software.name

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Structure visualization

Movie
  • 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-3185
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  • Superimposition on EM map
  • EMDB-3185
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: MAJOR INNER PROTEIN P1
B: MAJOR INNER PROTEIN P1


Theoretical massNumber of molelcules
Total (without water)168,3272
Polymers168,3272
Non-polymers00
Water0
1
A: MAJOR INNER PROTEIN P1
B: MAJOR INNER PROTEIN P1
x 60


Theoretical massNumber of molelcules
Total (without water)10,099,641120
Polymers10,099,641120
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation60
2


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3
A: MAJOR INNER PROTEIN P1
B: MAJOR INNER PROTEIN P1
x 5


  • icosahedral pentamer
  • 842 kDa, 10 polymers
Theoretical massNumber of molelcules
Total (without water)841,63710
Polymers841,63710
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation5
4
A: MAJOR INNER PROTEIN P1
B: MAJOR INNER PROTEIN P1
x 6


  • icosahedral 23 hexamer
  • 1.01 MDa, 12 polymers
Theoretical massNumber of molelcules
Total (without water)1,009,96412
Polymers1,009,96412
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation6
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 MAJOR INNER PROTEIN P1 / P1 PROTEIN FROM BACTERIOPHAGE PHI6


Mass: 84163.672 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) PSEUDOMONAS PHAGE PHI6 (bacteriophage) / Plasmid: PLM358 / Production host: PSEUDOMONAS SYRINGAE (bacteria) / References: UniProt: P11126

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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

ComponentName: BACTERIOPHAGE PHI6 POLYMERASE COMPLEX ASSEMBLED IN VITRO FROM PURIFIED PROTEINS P1, P2, AND P4
Type: COMPLEX
Buffer solutionName: 50 MM TRIS / pH: 8 / Details: 50 MM TRIS
SpecimenConc.: 2.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE
Details: VITRIFICATION 1 -- CRYOGEN- ETHANE, TEMPERATURE- 120, INSTRUMENT- FEI VITROBOT MARK IV, METHOD- BLOT 4 SECONDS BEFORE PLUNGING,

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F30 / Date: Jun 12, 2014 / Details: DOSE RATE 6-8 E- PER PIX PER SEC
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 160000 X / Calibrated magnification: 37037 X / Nominal defocus max: 2600 nm / Nominal defocus min: 1100 nm / Cs: 2 mm
Specimen holderTemperature: 81 K
Image recordingElectron dose: 16 e/Å2 / Film or detector model: GATAN K2 (4k x 4k)
Image scansNum. digital images: 834

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Processing

EM software
IDNameCategory
1Cootmodel fitting
2PHENIXmodel fitting
3UCSF Chimeramodel fitting
4RELION3D reconstruction
CTF correctionDetails: EACH PARTICLE
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionMethod: TEMPLATE BASED / Resolution: 4.8 Å / Num. of particles: 4379 / Nominal pixel size: 1.3 Å / Actual pixel size: 1.35 Å / Magnification calibration: ATOMIC MODEL
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-3185. (DEPOSITION ID: 13852).
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Details: METHOD--RIGID BODY REFINEMENT PROTOCOL--X-RAY
Atomic model buildingPDB-ID: 4K7H
RefinementHighest resolution: 4.8 Å
Refinement stepCycle: LAST / Highest resolution: 4.8 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms11840 0 0 0 11840

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