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- PDB-5fj6: Structure of the P2 polymerase inside in vitro assembled bacterio... -

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Basic information

Entry
Database: PDB / ID: 5fj6
TitleStructure of the P2 polymerase inside in vitro assembled bacteriophage phi6 polymerase complex
ComponentsRNA-DIRECTED RNA POLYMERASE
KeywordsTRANSCRIPTION / BACTERIOPHAGE PHI6 / POLYMERASE COMPLEX / P2 / POLYMERASE
Function / homology
Function and homology information


RNA uridylyltransferase activity / virion component / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / RNA binding / metal ion binding
Similarity search - Function
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
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 / Category: em_software
Item: _em_software.fitting_id / _em_software.image_processing_id / _em_software.name
Revision 1.3May 8, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_conn / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-3186
  • Imaged by UCSF Chimera
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Assembly

Deposited unit
A: RNA-DIRECTED RNA POLYMERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)74,9582
Polymers74,9031
Non-polymers551
Water00
1


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

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Components

#1: Protein RNA-DIRECTED 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) / Production host: PSEUDOMONAS SYRINGAE (bacteria) / References: UniProt: P11124
#2: 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 FROM PURIFIED PROTEINS P1, P2, AND P4
Type: COMPLEX
Buffer solutionName: 20 MM TRIS / pH: 8 / Details: 20 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 FIELD / 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
1PHENIXmodel fitting
2UCSF Chimeramodel fitting
3RELION3D 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-3186. (DEPOSITION ID: 13857).
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Details: METHOD--RIGID BODY REFINEMENT PROTOCOL--X-RAY
Atomic model buildingPDB-ID: 1HHS

1hhs


Accession code: 1HHS / Source name: PDB / Type: experimental model
RefinementHighest resolution: 7.9 Å
Refinement stepCycle: LAST / Highest resolution: 7.9 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5265 0 1 0 5266

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