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Title:Structure of bacteriophage phi29 prohead particle with a double insertion mutation (U92,U93) in the pRNA
Authors:Zhao W, Saha M, Ke A, Morais MC, Jardine PJ, Grimes S
Sample:Bacteriophage phi29 prohead particle with a double insertion mutation (U92,U93) in the pRNA
Method:Single particle reconstruction (17.3 angstroms resolution)
Red flagLatest update:2013-08-21
Sample
Sample name: Bacteriophage phi29 prohead particle with a double insertion mutation (U92,U93) in the pRNA
Oligomeric state: The prohead is derived from a prolate icosahedron with T = 3, Q = 5 quasi-symmetry wherein a pentameric capsomer at one end of the particle is replaced by a complex including a dodecameric connector protein and a pentameric pRNA
Theoretical molecular weight of the sample: 12.4
Components:
ID Type Name Exp. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1virusBacillus phage phi29Bacillus phage phi29The phi29 prohead particle was treated with RNase to remove the WT pRNA, and then incubated with exogenously produced pRNA engineered to contain a double U92,U93 insertion mutation.
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.80.1 mg/mL25 mM Tris-HCl, 5 mM MgCl2, 50 mM NaClQuantifoil holey carbon on top of200 mesh copper grid, plasma cleaned
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE% KHOMEMADE PLUNGERBlot grid from behind the sample for ~4 seconds before plunging. ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
JEOL 2200FS200 kVFLOOD BEAMBRIGHT FIELD2.0 mm640 nm3000 nm6000060000FIELD EMISSION GUNGATAN ULTRASCAN 4000 (4k x 4k) mmObjective lens astigmatism was corrected at 100,000 times magnification

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
Side entry liquid nitrogen-cooled cryo specimen holderGATAN LIQUID NITROGEN°°JEOL eV K K K mrad20 e/Å209-MAR-2011
Processing
Protocol:Model-based projection methods for alignment and Fourier methods for reconstruction
Software:EMAN
CTF correction:Each micrograph
Number of particles:2134
Resolution by author:17.3 Å
Resolution method:FSC 0.5
Processing details:C5 symmetry was imposed in all stages of the reconstruction, with the 5-fold axis along Z. The final map was low-pass filtered at 14 angstrom units. The particles were selected using the model-based automatic particle selection implemented in EMAN, followed by manual deletion of bad particles and manual selection of missed particles.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
109 μm/pixel
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails