EMD-2048 Experiments and Validation

Cryo-EM structure of the UPF-EJC complex

Single particle reconstruction
Overview of EMD-2048
Sample name: Complex containing the NMD proteins UPF1, UPF2 and UPF3 bound to the exon junction complex (EJC)
Organism: Homo sapiens

Map parameters

Recommended contour level: 2.40 (author)
Number of grid points: 136 × 136 × 136
Voxel size: 2.20 × 2.20 × 2.20 Å
Minimum density: -6.438
Maximum density: 17.814
Average density: -0.029
Standard deviation: 0.966

Sample information

Sample name: Complex containing the NMD proteins UPF1, UPF2 and UPF3 bound to the exon junction complex (EJC)
Nucleic acid: (U)8 RNA
Proteins: Up-frameshift protein 1, Up-frameshift protein 2, Up-frameshift protein 3b, MAGO, Y14, Btz, eIF4AIII

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: EMAN, XMIPP, SPIDER, BSOFT
Number of particles: 85000
Reconstruction protocol: angular refinement
CTF correction: Each CCD Frame using BSOFT
Details: Initial reference obtained by random conical tilt of negative-stained images

Fitting

Fitting 1
Initial atomic model: 1UW4
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: R-factor
Refinement space: REAL
Details: Protocol: Rigid body
Fitting 2
Initial atomic model: 2XB2
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: R-factor
Refinement space: REAL
Details: Protocol: Rigid body
Fitting 3
Initial atomic model: 2WJV
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: R-factor
Refinement space: REAL
Details: Protocol: Rigid body

Imaging

Session
Microscope model: JEOL 2200FS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 15.0
Energy filter: FEI
Energy window (eV): 0.00
Nominal CS (mm): 2.00
Nominal magnification: 50000.0
Defocus max (nm): 4,000.00
Defocus min (nm): 1,112.00
Holder model: GATAN LIQUID NITROGEN
Acquisition
Sampling interval (μm): 15.0
Details: 0.22 nm per pixel, final sampling

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.175
Buffer: 50 mM K-phosphate,150 mM NaCl, 3 mM MgCl2, 20% sucrose, 0.1% glutaraldehyde
Specimen support: Quantifoil grids (R2/2) with thin carbon film on top
Vitrification
Apparatus: FEI VITROBOT MARK III
Cryogen: ETHANE
Humidity: 95
Protocol: 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