EMD-5389 Experiments and Validation

An RNA Degradation Machine Sculpted by Ro Autoantigen and Noncoding RNA

Single particle reconstruction
Overview of EMD-5389
Sample name: Rsr/Y RNA/PNPase complex
Organism: Deinococcus radiodurans

Map parameters

Minimum density: -3.973
Maximum density: 14.191
Average density: 0.00
Standard deviation: 1.00
Recommended contour level: 3.67 (author)

Sample information

Sample name: Rsr/Y RNA/PNPase complex
Nucleic acid: Y RNA
Proteins: Ro sixty-related, polynucleotide phosphorylase

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: LEGINON, EMAN2, SPARX
Number of particles: 9000
Number of class averages: 50
Reconstruction protocol: RCT followed by projection matching refinement
CTF correction: Each particle in IMAGIC

Fitting

Fitting 1
Initial atomic model: 1E3P
Used chain: A
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: Cross-correlation coefficient
Refinement space: REAL
Details: Protocol: Rigid body using Fit-in-Map. A trimer of the crystal structure of PNPase was created using COOT and used for fitting.
Fitting 2
Initial atomic model: 1YVP
Used chains: B, E, F, H
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: Cross-correlation coefficient
Refinement space: REAL
Details: Protocol: Rigid body using Fit-in-Map. Combined with the double-stranded portion of the misfolded substrate RNA from PDB 2I91.
Fitting 3
Initial atomic model: 2I91
Used chains: C, D
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: Cross-correlation coefficient
Refinement space: REAL
Details: Protocol: 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.

Imaging

Session
Microscope model: FEI TECNAI 12
Electron source: LAB6
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.30
Nominal magnification: 49000.0
Defocus max (nm): 1,200.00
Defocus min (nm): 800.00
Holder model: OTHER
Acquisition
Number of images: 40

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.10
Buffer: 20 mM HEPES, 50 mM NaCl, 2 mM beta-mercaptoethanol, 1 mM MgCl2, 1 mM MnCl2, 1 mM Petabloc
Staining procedure: Grids with adsorbed protein were stained consecutively with 3 droplets of 2% w/v uranyl acetate for 10 seconds each.
Specimen support: Homemade holey carbon grids with a thin layer of carbon over the holes
Vitrification
Apparatus: NONE
Cryogen: NONE