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Title:CryoEM reconstruction of the bacteriophage phi6 procapsid to the near-atomic resolution
Authors:Nemecek D, Boura E, Wu W, Cheng N, Plevka P, Qiao J, Mindich L, Heymann JB, Hurley JH, Steven AC
Sample:Wildtype P1247 procapsid of bacteriophage phi6
Method:Single particle reconstruction (4.4 angstroms resolution)
Red flagLatest update:2013-08-21
Sample
Sample name: Wildtype P1247 procapsid of bacteriophage phi6
Oligomeric state: icosahedral shell with accessory proteins
Theoretical molecular weight of the sample: 12.6
Components:
ID Type Name Exp. MW (MDa) Theo. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1virusPseudomonas phage phi612.6Pseudomonas phage phi6
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
8.010 mg/mL10 mM potassium phosphate, 5 mM MgCl2400 mesh C-flat holey carbon grid
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE80%100 KFEI VITROBOT MARK IBlot for 2 seconds before plunging. ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TITAN KRIOS300 kVFLOOD BEAMBRIGHT FIELD2.7 mm800 nm2200 nm4600044739FIELD EMISSION GUNKODAK SO-163 FILM mm

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
FEI TITAN KRIOS AUTOGRID HOLDER eV K K K mrad15 e/Å220-NOV-2011
Processing
Software:EMAN, BSOFT
CTF correction:Particles from each micrograph
Number of particles:18326
Imposed symmetry:I
Resolution by author:4.4 Å
Resolution method:FSC 0.5
Processing details:The particles were selected using e2boxer (EMAN) and manually pruned in bshow (BSOFT). The initial model was taken from our previous reconstruction at 7A resolution (EMD-2341). The final structure was reconstructed in EMAN.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
2206.35 μm/pixel16NIKON SUPER COOLSCAN 9000
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
4K7H flexibleChimera, MDFF, CootREALthe P1 crystal structure was rigid body-fitted into the procapsid map using Chimera. Regions where the crystal structure deviated significantly from the EM density were roughly adjusted in Coot and then the P1A and P1B structures were flexibly fitted using the MDFF package. Finally, both structures were visually inspected and refined in Coot.