EMD-5604 Experiments and Validation

Substrate-specific structural rearrangements of human Dicer

Single particle reconstruction
Overview of EMD-5604
Sample name: Human Dicer-PACT heterodimer in closed conformation
Organism: Homo sapiens
Related EM entries by publication: EMD-5605, EMD-5606, EMD-5601, EMD-5603, EMD-5602

Map parameters

Minimum density: -5.237
Maximum density: 19.375
Average density: 0.00
Standard deviation: 1.00
Recommended contour level: 4.58 (author)

Sample information

Sample name: Human Dicer-PACT heterodimer in closed conformation
Proteins: Endoribonuclease Dicer, Interferon-inducible double stranded RNA-dependent protein kinase activator A

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: EMAN2/SPARX, multi-model
Number of particles: 10000
Reconstruction protocol: back projection
CTF correction: each micrograph

Imaging

Session
Microscope model: FEI TECNAI 12
Electron source: LAB6
Electron dose (e/A**2): 20.0
Nominal CS (mm): 5.20
Nominal magnification: 50000.0
Defocus max (nm): 1,500.00
Defocus min (nm): 400.00
Holder model: OTHER
Acquisition
Number of images: 400

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.01
Buffer: 20 mM HEPES, pH 7.5, 150 mM KCl, 3 mM EDTA, 1 mM DTT, and 2.5% glycerol
Staining procedure: After adsorption for 1 min, we stained the samples consecutively with three droplets of 2% (w/v) uranyl formate solution, blotted off the residual stain and air-dried the sample in a hood.
Specimen support: glow-discharged holey carbon grids with a thin layer of carbon over the holes
Vitrification
Apparatus: NONE
Cryogen: NONE