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- EMDB-3187: Structure of the P2 polymerase inside in vitro assembled bacterio... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-3187 | |||||||||
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Title | Structure of the P2 polymerase inside in vitro assembled bacteriophage phi6 polymerase complex, with P1 included | |||||||||
![]() | Localized reconstruction of P2 polymerase from bacteriophage phi6 polymerase complexes assembled in vitro, together with P1 | |||||||||
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![]() | Bacteriophage phi6 / polymerase complex / P2 / polymerase / P1 | |||||||||
Function / homology | ![]() T=2 icosahedral viral capsid / viral procapsid / RNA uridylyltransferase activity / viral genome packaging / viral inner capsid / ribonucleoside triphosphate phosphatase activity / virion component / viral capsid / nucleoside-triphosphate phosphatase / viral nucleocapsid ...T=2 icosahedral viral capsid / viral procapsid / RNA uridylyltransferase activity / viral genome packaging / viral inner capsid / ribonucleoside triphosphate phosphatase activity / virion component / viral capsid / nucleoside-triphosphate phosphatase / viral nucleocapsid / RNA-directed RNA polymerase / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / RNA binding / ATP binding / identical protein binding / metal ion binding Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 7.9 Å | |||||||||
![]() | Ilca S / Kotecha A / Sun X / Poranen MP / Stuart DI / Huiskonen JT | |||||||||
![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 3.4 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 13.5 KB 13.5 KB | Display Display | ![]() |
Images | ![]() ![]() | 173.4 KB 173.4 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 5fj7MC ![]() 3183C ![]() 3184C ![]() 3185C ![]() 3186C ![]() 5fj5C ![]() 5fj6C M: atomic model generated by this map C: citing same article ( |
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Similar structure data | |
EM raw data | ![]() Data size: 8.1 Data #1: Extracted particle stacks of bacteriophage phi6 polymerase complex [picked particles - multiframe - processed]) |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Localized reconstruction of P2 polymerase from bacteriophage phi6 polymerase complexes assembled in vitro, together with P1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.35 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Bacteriophage phi6 polymerase complex assembled in vitro from pur...
Entire | Name: Bacteriophage phi6 polymerase complex assembled in vitro from purified proteins P1, P2, and P4 |
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Components |
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-Supramolecule #1000: Bacteriophage phi6 polymerase complex assembled in vitro from pur...
Supramolecule | Name: Bacteriophage phi6 polymerase complex assembled in vitro from purified proteins P1, P2, and P4 type: sample / ID: 1000 Oligomeric state: Icosahedral assembly with 120 copies of P1 Number unique components: 3 |
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-Macromolecule #1: P1 protein from bacteriophage phi6
Macromolecule | Name: P1 protein from bacteriophage phi6 / type: protein_or_peptide / ID: 1 / Number of copies: 120 Oligomeric state: 60 asymmetric dimers from an icosahedral shell Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 85 KDa |
Recombinant expression | Organism: ![]() |
Sequence | UniProtKB: Major inner protein P1 |
-Macromolecule #2: P2 protein from bacteriophage phi6
Macromolecule | Name: P2 protein from bacteriophage phi6 / type: protein_or_peptide / ID: 2 / Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 75 KDa |
Recombinant expression | Organism: ![]() |
Sequence | UniProtKB: RNA-directed RNA polymerase |
-Macromolecule #3: P4 protein from bacteriophage phi6
Macromolecule | Name: P4 protein from bacteriophage phi6 / type: protein_or_peptide / ID: 3 / Oligomeric state: hexamer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 35 KDa |
Recombinant expression | Organism: ![]() |
Sequence | UniProtKB: Packaging enzyme P4 |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 2.4 mg/mL |
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Buffer | pH: 8 / Details: 20 mM Tris |
Grid | Details: glow discharged Cflat grid (CF-2/1-2C-T) |
Vitrification | Cryogen name: ETHANE / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK IV / Method: Blot 4 seconds before plunging |
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Electron microscopy
Microscope | FEI POLARA 300 |
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Temperature | Min: 81 K / Max: 120 K / Average: 81 K |
Specialist optics | Energy filter - Name: GIF QUANTUM LS / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV |
Details | dose rate 6-8 e-/pix/s |
Date | Jun 12, 2014 |
Image recording | Category: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 834 / Average electron dose: 16 e/Å2 Details: Every image is the average of 22 frames recorded by the direct electron detector Bits/pixel: 16 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Calibrated magnification: 37037 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 160000 |
Sample stage | Specimen holder model: OTHER |
Experimental equipment | ![]() Model: Tecnai Polara / Image courtesy: FEI Company |
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Image processing
Details | After 3D classification, particles from the classes that contained P2 were selected |
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CTF correction | Details: Each particle |
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.9 Å / Resolution method: OTHER / Software - Name: Relion Details: Final map was postprocessed in Relion. Inverse B-factor of 515 was applied. Number images used: 43216 |
Final two d classification | Number classes: 5 |
-Atomic model buiding 1
Initial model | PDB ID: Chain - Chain ID: A |
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Software | Name: Chimera, COOT, Phenix |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
Output model | ![]() PDB-5fj7: |