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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) Latest update: 2012-08-29   Summary  Experimental details   Visualization   Map information   Downloads Sample Sample name: Complex containing the NMD proteins UPF1, UPF2 and UPF3 bound to the exon junction complex (EJC) Oligomeric state: Hetero-octameric Theoretical molecular weight of the sample: 0.390 Components: ID Type Name Exp. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details 1 protein Up-frameshift protein 1 Monomer true Homo sapiens GO:0000184 IPR018999 115-914 2 protein Up-frameshift protein 2 Monomer true Homo sapiens GO:0000184 IPR018999 full length 3 protein Up-frameshift protein 3b Monomer true Homo sapiens GO:0000184 IPR005120 full length 4 protein MAGO Monomer true Homo sapiens GO:0000184 IPR015362 full length 5 protein Y14 Monomer true Homo sapiens GO:0000184 IPR015362 residues 66-154 6 protein Btz Monomer true Homo sapiens GO:0000184 IPR018545 residues 136-286 7 protein eIF4AIII Monomer true Homo sapiens GO:0000184 IPR018545 full length 8 nucleic-acid (U)8 RNA true Homo sapiens Oligo(U) 8nt Experiment Specimen state: Particle Specimen preparation: pH Specimen conc. Details Staining Specimen support details 7.5 0.175 mg/mL 50 mM K-phosphate,150 mM NaCl, 3 mM MgCl2, 20% sucrose, 0.1% glutaraldehyde Quantifoil grids (R2/2) with thin carbon film on top Vitrification: Cryogen name Humidity Temp. Instr. Method Time resolved Details ETHANE 95% K FEI VITROBOT MARK III Manual 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: Microscope Voltage Illumination mode Imaging mode Cs Defocus min. Defocus max. Nominal mag. Calibrated mag. Electron source Detector Detector distance Astigmatism JEOL 2200FS 200 kV FLOOD BEAM BRIGHT FIELD 2.0 mm 1112 nm 4000 nm 50000 69494 FIELD EMISSION GUN ULTRASCAN 4000 SP GATAN slow-scan CCD camera mm Objective lens astigmatism was corrected calculating the Fourier Transform of the CCD frames. Phase flipping Specimen holder Holder model Tilt min. Tilt max. Energy filter Energy window Temp. Temp. min. Temp. max. Beam tilt Electron dose Other details Date Eucentric. 626 cryo-holder (Gatan Inc., Warrendale, PA, USA) GATAN LIQUID NITROGEN ° ° FEI 0-10 eV 91.5 K 83 K 100 K mrad 15 e/Å2 20-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 at 0.5 cut-off Processing details: Initial reference obtained by random conical tilt of negative-stained images. Scanned images: Num. images Sampling size OD range Quant. bit number Other details Scanner 15 μm/pixel 16 0.22 nm per pixel, final sampling Fitting: PDB Protocol Target crit. Software B value Fitting space PDB chain Details 1UW4  rigid body R-factor Chimera REAL Protocol: Rigid body 2XB2  rigid body R-factor Chimera REAL Protocol: Rigid body 2WJV  rigid body R-factor Chimera REAL Protocol: Rigid body