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Title:An RNA Degradation Machine Sculpted by Ro Autoantigen and Noncoding RNA
Authors:Chen X, Taylor DW, Wang HW, Wolin SL
Sample:Rsr/Y RNA/PNPase complex
Method:Single particle reconstruction (25 angstroms resolution)
Red flagLatest update:2013-04-10
Sample
Sample name: Rsr/Y RNA/PNPase complex
Oligomeric state: one Rsr-Y RNA complex binds to one PNPase trimer
Theoretical molecular weight of the sample: 0.365
Components:
ID Type Name Exp. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1proteinRo sixty-relatedmonomertrueDeinococcus radioduransHis-tag Rsr
2nucleic-acidY RNAfalseDeinococcus radioduransY RNA generated with correct 3' end using hammerhead ribozyme
3proteinpolynucleotide phosphorylaseTrimertrueDeinococcus radioduransStrep-tag PNPase
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.50.1 mg/mL20 mM HEPES, 50 mM NaCl, 2 mM beta-mercaptoethanol, 1 mM MgCl2, 1 mM MnCl2, 1 mM PetablocGrids with adsorbed protein were stained consecutively with 3 droplets of 2% w/v uranyl acetate for 10 seconds each.Homemade holey carbon grids with a thin layer of carbon over the holes
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
NONE% KNONE ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TECNAI 12120 kVFLOOD BEAMBRIGHT FIELD2.3 mm800 nm1200 nm49000LAB6TVIPS TEMCAM-F416 (4k x 4k) mmobject lens astigmatism was corrected at 42,000 magnification

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
OTHER°° eV K K K mrad20 e/Å212-JUL-2009
Processing
Protocol:RCT followed by projection matching refinement
Software:LEGINON, EMAN2, SPARX
CTF correction:Each particle in IMAGIC
Number of particles:9000
Number of class averages:50
Imposed symmetry:C1
Resolution by author:25 Å
Resolution method:FSC 0.5
Processing details:The particles were selected manually using boxer in EMAN.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
40 μm/pixel
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
1E3P rigid bodyCross-correlation coefficientChimeraREALProtocol: Rigid body using Fit-in-Map. A trimer of the crystal structure of PNPase was created using COOT and used for fitting.
1YVP rigid bodyCross-correlation coefficientChimeraREALProtocol: Rigid body using Fit-in-Map. Combined with the double-stranded portion of the misfolded substrate RNA from PDB 2I91.
2I91 rigid bodyCross-correlation coefficientChimeraREALProtocol: Rigid body using Fit-in-Map. The double-stranded portion of the misfolded substrate RNA from this structure was combined with PDB 1YVP to create a model of Rsr bound by Y RNA and a full substrate RNA with single-stranded tail.