Journal: J Virol / Year: 2012 Title: Packaging accessory protein P7 and polymerase P2 have mutually occluding binding sites inside the bacteriophage 6 procapsid. Authors: Daniel Nemecek / Jian Qiao / Leonard Mindich / Alasdair C Steven / J Bernard Heymann / Abstract: Bacteriophage 6 is a double-stranded RNA (dsRNA) virus whose genome is packaged sequentially as three single-stranded RNA (ssRNA) segments into an icosahedral procapsid which serves as a compartment ...Bacteriophage 6 is a double-stranded RNA (dsRNA) virus whose genome is packaged sequentially as three single-stranded RNA (ssRNA) segments into an icosahedral procapsid which serves as a compartment for genome replication and transcription. The procapsid shell consists of 60 copies each of P1(A) and P1(B), two nonequivalent conformers of the P1 protein. Hexamers of the packaging ATPase P4 are mounted over the 5-fold vertices, and monomers of the RNA-dependent RNA polymerase (P2) attach to the inner surface, near the 3-fold axes. A fourth protein, P7, is needed for packaging and also promotes assembly. We used cryo-electron microscopy to localize P7 by difference mapping of procapsids with different protein compositions. We found that P7 resides on the interior surface of the P1 shell and appears to be monomeric. Its binding sites are arranged around the 3-fold axes, straddling the interface between two P1(A) subunits. Thus, P7 may promote assembly by stabilizing an initiation complex. Only about 20% of the 60 P7 binding sites were occupied in our preparations. P7 density overlaps P2 density similarly mapped, implying mutual occlusion. The known structure of the 12 homolog fits snugly into the P7 density. Both termini-which have been implicated in RNA binding-are oriented toward the adjacent 5-fold vertex, the entry pathway of ssRNA segments. Thus, P7 may promote packaging either by interacting directly with incoming RNA or by modulating the structure of the translocation pore.
History
Deposition
Mar 27, 2013
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Header (metadata) release
Apr 10, 2013
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Map release
Apr 10, 2013
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Update
Apr 10, 2013
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Current status
Apr 10, 2013
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Shell ID: 1 / Diameter: 450 Å / T number (triangulation number): 2
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
10 mg/mL
Buffer
pH: 8 / Details: 10mM Tris, 5mM MgCl2
Grid
Details: Q-foil 2/2/300 mesh copper grid
Vitrification
Cryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK I / Method: Blot for 2 seconds before plunging
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Electron microscopy
Microscope
FEI/PHILIPS CM200FEG
Electron beam
Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.35 µm / Number real images: 4047 / Average electron dose: 15 e/Å2 / Bits/pixel: 16
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Image processing
CTF correction
Details: CTF was determined from whole micrographs
Final reconstruction
Applied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 12.4 Å / Resolution method: FSC 0.33 CUT-OFF / Software - Name: Bsoft / Number images used: 4047
Details
particles were selected manually, CTF was determined from whole micrographs
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