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- PDB-5fj7: Structure of the P2 polymerase inside in vitro assembled bacterio... -

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Basic information

Entry
Database: PDB / ID: 5fj7
TitleStructure of the P2 polymerase inside in vitro assembled bacteriophage phi6 polymerase complex, with P1 included
Components
  • MAJOR INNER PROTEIN P1
  • RNA-DIRECTED RNA POLYMERASERNA-dependent RNA polymerase
KeywordsVIRAL PROTEIN / BACTERIOPHAGE PHI6 / POLYMERASE COMPLEX / P2 / POLYMERASE / P1
Function / homology
Function and homology information


T=2 icosahedral viral capsid / RNA uridylyltransferase activity / viral inner capsid / virion component / viral nucleocapsid / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription ...T=2 icosahedral viral capsid / RNA uridylyltransferase activity / viral inner capsid / virion component / viral nucleocapsid / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / RNA binding / metal ion binding / identical protein binding
Similarity search - Function
: / Major inner capsid protein P1 / Cystovirus, RNA-directed RNA polymerase, N-terminal / Cystovirus, RNA-directed RNA polymerase / RNA-directed RNA polymerase, bacteriophage, catalytic domain / RdRp of RNA-containing bacteriophages catalytic domain profile. / RNA-directed RNA polymerase, C-terminal domain / Viral RNA-dependent RNA polymerase / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
: / RNA-directed RNA polymerase / Major inner protein P1
Similarity search - Component
Biological speciesPSEUDOMONAS PHAGE PHI6 (bacteriophage)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.9 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.
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 / Refinement description / Category: em_3d_fitting / em_software
Item: _em_3d_fitting.target_criteria / _em_software.fitting_id ..._em_3d_fitting.target_criteria / _em_software.fitting_id / _em_software.image_processing_id / _em_software.name
Revision 1.3Nov 20, 2019Group: Derived calculations / Category: pdbx_struct_conn_angle / struct_conn

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Structure visualization

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  • EMDB-3187
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Assembly

Deposited unit
A: MAJOR INNER PROTEIN P1
B: MAJOR INNER PROTEIN P1
C: RNA-DIRECTED RNA POLYMERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)243,2854
Polymers243,2313
Non-polymers551
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA

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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
#2: Protein RNA-DIRECTED RNA POLYMERASE / RNA-dependent RNA polymerase / PROTEIN P2 / P2 PROTEIN FROM BACTERIOPHAGE PHI6


Mass: 74903.203 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) PSEUDOMONAS PHAGE PHI6 (bacteriophage) / Plasmid: PLM358 / Production host: PSEUDOMONAS SYRINGAE (bacteria) / References: UniProt: P11124
#3: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn

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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
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 S
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: 0.16 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: C1 (asymmetric)
3D reconstructionMethod: LOCALIZED RECONSTRUCTION / Resolution: 7.9 Å / Num. of particles: 43216 / 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-3187. (DEPOSITION ID: 13864).
Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL / Target criteria: Cross-correlation coefficient
Details: METHOD--LOCAL CORRELATION REFINEMENT PROTOCOL--X-RAY
Atomic model building
IDPDB-ID 3D fitting-ID
14K7H1
21HHS1
RefinementHighest resolution: 7.9 Å
Refinement stepCycle: LAST / Highest resolution: 7.9 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17105 0 1 0 17106

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