EMD-2304 Experiments and Validation

Structural visualization of key steps in human transcription initiation

Single particle reconstruction
Overview of EMD-2304
Sample name: TBP-TFIIA-TFIIB-DNA-Pol II (close)
Organism: Homo sapiens
Related EM entries by publication: EMD-2309, EMD-2308, EMD-2305, EMD-2307, EMD-2306

Map parameters

Minimum density: -10.48
Maximum density: 16.48
Average density: 0.01
Standard deviation: 0.67
Recommended contour level: 2.5 (author)

Sample information

Sample name: TBP-TFIIA-TFIIB-DNA-Pol II (close)
Nucleic acid: Core promoter DNA
Proteins: TATA-box-binding protein, General transcription factor IIA, General transcription factor IIB, DNA-directed RNA polymerase II

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: EMAN2, SPARX
Number of particles: 122480
Reconstruction protocol: Projection matching
CTF correction: whole micrograph
Details: Image processing was performed in the Appion processing environment. 3D reconstruction was performed using EMAN2 and SPARX libraries. Final map was filtered to local resolution using the blocres function in Bsoft package.

Imaging

Session
Microscope model: FEI TECNAI F20
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.2
Nominal magnification: 100000.0
Defocus max (nm): 2,400
Defocus min (nm): 1,200
Holder model: GATAN LIQUID NITROGEN
Details: Data acquired using Leginon
Acquisition
Number of images: 3000

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.07
Buffer: 10 mM HEPES, 5% glycerol, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.05% NP-40
Specimen support: 400-mesh C-flats with thin carbon support over 4um holes with 2um spacing (Protochips Inc.)
Vitrification
Apparatus: FEI VITROBOT MARK II
Cryogen: ETHANE
Humidity: 100
Protocol: Incubation on grid for 5 minutes before blotting 4 seconds at -1 offset.