EMD-5801 Experiments and Validation

Structure of the mammalian ribosomal pre-termination complex associated with eRF1-eRF3-GDPNP

Single particle reconstruction
Overview of EMD-5801
Sample name: eRF1-eRF3-GDPNP-bound mammalian ribosomal pre-termination complex
Organisms: Oryctolagus cuniculus, Homo sapiens
Fitted atomic model: 3j5y

Map parameters

Recommended contour level: 0.02 (author)
Number of grid points: 304 × 304 × 304
Voxel size: 1.50 × 1.50 × 1.50 Å
Minimum density: -0.025
Maximum density: 0.081
Average density: 0.001
Standard deviation: 0.009

Sample information

Sample name: eRF1-eRF3-GDPNP-bound mammalian ribosomal pre-termination complex
Nucleic acids: Transfer-messenger RNA Leu, messenger RNA
Proteins: eukaryotic release factor 1, eukaryotic release factor 3
Ribosome: 80S ribosome

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: RELION
Number of particles: 48973
Reconstruction protocol: reference-based reconstruction
CTF correction: Each Particle

Fitting

Fitting 1
Initial atomic model: 3E1Y
Used chains: A, F
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: Cross correlation
Refinement space: REAL
Details: Domains were fitted as rigid bodies using Chimera. Individual domains were then refined manually using hinge points as points of flexibility.
Fitting 2
Initial atomic model: 2KTU
Used chain: A
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: Cross correlation
Refinement space: REAL
Fitting 3
Initial atomic model: 1R5B
Used chain: A
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: Cross correlation
Refinement space: REAL
Fitting 4
Initial atomic model: 1R5O
Used chain: A
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: Cross correlation
Refinement space: REAL
Fitting 5
Initial atomic model: 3VMF
Used chain: A
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: Cross correlation
Refinement space: REAL

Imaging

Session
Microscope model: FEI POLARA 300
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 25.0
Nominal CS (mm): 2.10
Holder model: GATAN HELIUM

Specimen

Preparation
Specimen state: particle
Buffer: 20 mM Tris, 100 mM KOAc, 2.5 mM MgCl2, 2 mM DTT, 0.25 mM spermidine supplemented with 200 units RNasin, 0.4 mM ATP, 3 mM Mg-GMPPNP
Staining procedure: cryo
Specimen support: Quantifoil grids, glow discharged
Vitrification
Apparatus: FEI VITROBOT MARK IV
Cryogen: ETHANE
Humidity: 100