EMD-2287 Experiments and Validation

Human TFIID binds to core promoter DNA in a reorganized structural state

Single particle reconstruction
Overview of EMD-2287
Sample name: holo-TFIID (canonical)
Organism: Homo sapiens
Related EM entries by publication: EMD-2282, EMD-2283, EMD-2284

Map parameters

Minimum density: -0.11
Maximum density: 0.71
Average density: -0.01
Standard deviation: 0.03
Recommended contour level: 0.13 (author)

Sample information

Sample name: holo-TFIID (canonical)
Protein: Transcription factor II-D

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: Sparx, EMAN2, FREALIGN
Number of particles: 11898
Reconstruction protocol: Common lines
CTF correction: Per micrograph for phase flipping, per particles for refinement in FREALIGN

Imaging

Session
Microscope model: FEI TECNAI F20
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 25.0
Nominal CS (mm): 2.2
Nominal magnification: 80000.0
Defocus max (nm): 4,500
Defocus min (nm): 1,800
Holder model: SIDE ENTRY, EUCENTRIC
Acquisition
Number of images: 494
Scanner model: OTHER

Specimen

Preparation
Specimen concentration (mg/mL): 0.2
Buffer: 20 mM HEPES pH 7.8, 0.2 mM EDTA, 4 mM MgCl2, ~1% glycerol, 1 mM DTT, 3% trehalose, 50 mM KCl, 0.05% NP-40,
Specimen support: 400 mesh copper grid on C-flat support carbon with a thin carbon support over 4 um holes space 2 um apart.
Vitrification
Apparatus: FEI VITROBOT MARK II
Cryogen: ETHANE
Humidity: 100
Protocol: Sample was incubated on grid for 30 seconds before blotting 6.5s at -1 offset. The grid was washed in 4 ul buffer to blot again for 6.5 s at -1 offset prior to plunging.