EMD-2511 Experiments and Validation

Cryo-EM of the Sec61-complex bound to an 80S-RNC engaged with a translocating LepT chain

Single particle reconstruction
Overview of EMD-2511
Sample name: Canis familiaris Sec61 bound to a wheat germ 80S-RNC translating the translocating LepT-polypeptide
Organisms: Canis lupus familiaris, Triticum aestivum
Fitted atomic model: 4cg5
Related EM entry by publication: EMD-2510, EMD-2512

Map parameters

Recommended contour level: 0.10 (author)
Number of grid points: 368 × 368 × 368
Voxel size: 1.24 × 1.24 × 1.24 Å
Minimum density: -0.649
Maximum density: 1.033
Average density: 0.007
Standard deviation: 0.062

Sample information

Sample name: Canis familiaris Sec61 bound to a wheat germ 80S-RNC translating the translocating LepT-polypeptide
Protein: Sec61-complex
Ribosome: 80S Ribosome

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: SPIDER, Signature, MAPPOS
Number of particles: 53248
CTF correction: On 3D-volume (SPIDER)

Fitting

Initial atomic model: 2WWB
Fitting software: Chimera, Coot, MDFF
Fitting protocol: flexible
Refinement space: REAL

Imaging

Session
Microscope model: FEI TITAN KRIOS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 25.0
Nominal CS (mm): 2.70
Defocus max (nm): 4,000.00
Defocus min (nm): 1,300.00
Holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Acquisition
Number of images: 9805

Specimen

Preparation
Specimen state: particle
Buffer: 30 mM Hepes/KOH 7.6, 10 mM Mg(OAc)2, 180 mM KOAC/HAc pH 7.6, 0.3 % Digitonin, 1 mM DTT
Specimen support: Quantifoil grids pre-coated with 2 nm carbon on top
Vitrification
Apparatus: FEI VITROBOT MARK IV
Cryogen: ETHANE
Humidity: 95
Protocol: Blot for 3 seconds before plunging