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Title:Cryo-EM structure of the UPF-EJC complex
Authors:Melero R, Buchwald G, Castano R, Raabe M, Gil D, Lazaro M, Urlaub H, Conti E, Llorca O
Sample:Complex containing the NMD proteins UPF1, UPF2 and UPF3 bound to the exon junction complex (EJC)
Method:Single particle reconstruction (16 angstroms resolution)
Red flagLatest update:2012-08-29
Sample
Sample name: Complex containing the NMD proteins UPF1, UPF2 and UPF3 bound to the exon junction complex (EJC)
Oligomeric state: Hetero-octameric
Experimental molecular weight of the sample: 0.390
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
1proteinUp-frameshift protein 10.090MonomertrueHomo sapiensGO:0000184IPR018999115-914
2proteinUp-frameshift protein 20.148MonomertrueHomo sapiensGO:0000184IPR018999full length
3proteinUp-frameshift protein 3b0.056MonomertrueHomo sapiensGO:0000184IPR005120full length
4proteinMAGO0.016MonomertrueHomo sapiensGO:0000184IPR015362full length
5proteinY140.010MonomertrueHomo sapiensGO:0000184IPR015362residues 66-154
6proteinBtz0.017MonomertrueHomo sapiensGO:0000184IPR018545residues 136-286
7proteineIF4AIII0.045MonomertrueHomo sapiensGO:0000184IPR018545full length
8nucleic-acid(U)8 RNA0.003trueHomo sapiensOligo(U) 8nt
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.50.175 mg/mL50 mM K-phosphate,150 mM NaCl, 3 mM MgCl2, 20% sucrose, 0.1% glutaraldehydeQuantifoil grids (R2/2) with thin carbon film on top
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE95% KFEI VITROBOT MARK IIIManual Application (3.5 microliters) Humidifier Off During Process Blot offset: -2 mm Blot Total: 2 Blot Time: 2 s Wait Time: 30 s Drain Time: 1 s ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
JEOL 2200FS200 kVFLOOD BEAMBRIGHT FIELD2.0 mm1112 nm4000 nm5000069494FIELD EMISSION GUNGATAN ULTRASCAN 4000 (4k x 4k) mmObjective lens astigmatism was corrected calculating the Fourier Transform of the CCD frames. Phase flipping

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
Eucentric. 626 cryo-holder (Gatan Inc., Warrendale, PA, USA)GATAN LIQUID NITROGEN°°FEI0-10 eV91.5 K83 K100 K mrad15 e/Å220-OCT-2010
Processing
Protocol:angular refinement
Software:EMAN, XMIPP, SPIDER, BSOFT
CTF correction:Each CCD Frame using BSOFT
Number of particles:85000
Resolution by author:16 Å
Resolution method:FSC 0.5
Processing details:Initial reference obtained by random conical tilt of negative-stained images.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
15 μm/pixel160.22 nm per pixel, final sampling
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
1UW4 rigid bodyR-factorChimeraREALProtocol: Rigid body
2XB2 rigid bodyR-factorChimeraREALProtocol: Rigid body
2WJV rigid bodyR-factorChimeraREALProtocol: Rigid body