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Title:Reconstruction of the deltaGCC mutant HCV IRES bound to rabbit 40S ribosomal subunit
Authors:Vollmar BS, Shi D, Filbin ME, Kieft JS, Gonen T
Sample:mutant HCV IRES bound to 40S
Method:Single particle reconstruction (17.5 angstroms resolution)
Sample
Sample name: mutant HCV IRES bound to 40S
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
1ribosome-eukaryoteRabbit 40S ribosomal subunitfalseOryctolagus cuniculusMutant HCV IRES bound to 40S
2nucleic-acidhepatitis C virus internal ribosome entry sitetrueHepatitis C virusDeletion of GCC (82-84) of HCV IRES (nucleotides 40-372)
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.4 mg/mL20 mM Tris-HCl, pH 7.4, 200 mM KOAc, 2.5 mM MgCl2, 2 mM DTT, 40 mM KClQuantifoil R1.2/1.3 400 mesh copper grids with holey carbon
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE100% KFEI VITROBOT MARK IVBlot and plunge into liquid ethane ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TECNAI F20200 kVSPOT SCANBRIGHT FIELD2.0 mm1250 nm5000 nm62000117293FIELD EMISSION GUNGENERIC TVIPS (4k x 4k) mm

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
GATAN LIQUID NITROGEN eV K K K mrad15 e/Å219-MAR-2012
Processing
Software:FREALIGN
CTF correction:CTFFIND3
Number of particles:29920
Imposed symmetry:C1
Resolution by author:17.5 Å
Resolution method:RMEASURE
Processing details:Particles selected manually using Electron Micrographe Utility (cryoem.ucsf.edu).
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
1790 μm/pixelUsed 2x binned images yielding a pixel size of 2.66 A/pixel.
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails