EMD-2394 Experiments and Validation

Architecture of an RNA polymerase II transcription pre-initiation complex

Single particle reconstruction
Overview of EMD-2394
Sample name: RNA polymerase II transcription pre-initiation complex
Organism: Saccharomyces cerevisiae

Map parameters

Recommended contour level: 125,000.00 (emdb)
Number of grid points: 200 × 200 × 200
Voxel size: 1.85 × 1.85 × 1.85 Å
Minimum density: -836,502.80
Maximum density: 3,397,460.80
Average density: 16,963.73
Standard deviation: 128,061.195

Sample information

Sample name: RNA polymerase II transcription pre-initiation complex
Protein: RNA polymerase II transcription pre-initiation complex

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: SIMPLE
Number of particles: 433916
Reconstruction protocol: common lines
CTF correction: frame

Imaging

Session
Microscope model: FEI TECNAI F20
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 15.0
Nominal CS (mm): 2.26
Nominal magnification: 81000.0
Defocus max (nm): 3,500.00
Defocus min (nm): 1,500.00
Holder model: GATAN LIQUID NITROGEN

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.30
Buffer: 20mM HEPES, 5mM DTT, 2mM magnesium acetate, 30 mM potassium acetate
Staining procedure: 20 ul of fixed sample was dialyzed for 2 hours to remove glycerol. 3 ul were transferred to a grid and flash frozen in liquid ethane with a Vitrobot.
Specimen support: Quantifoil was glow-discharged using a Harrick Plasma Cleaner
Vitrification
Apparatus: FEI VITROBOT MARK III
Cryogen: ETHANE
Humidity: 100
Protocol: blot time 3 sec, waiting 10sec, force 9