EMD-2367 Experiments and Validation

Structural mimicry in transcription regulation of human RNA polymerase II by the DNA helicase RECQL5

Single particle reconstruction
Overview of EMD-2367
Sample name: Human Pol II in complex with artificial transcription bubble and RECQL5
Organisms: Homo sapiens, unidentified

Map parameters

Recommended contour level: 0.005 (author)
Number of grid points: 176 × 176 × 176
Voxel size: 2.31 × 2.31 × 2.31 Å
Minimum density: -0.036
Maximum density: 0.054
Average density: 0.00
Standard deviation: 0.003

Sample information

Sample name: Human Pol II in complex with artificial transcription bubble and RECQL5
Nucleic acids: transcription bubble RNA strand, non-template strand, template strand
Proteins: RNA polymerase II, RECQL5

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: EMAN2, SPARX
Number of particles: 121416
Reconstruction protocol: Projection matching
CTF correction: whole micrograph

Fitting

Fitting 1
Initial atomic model: 1Y1W
Fitting software: Chimera
Fitting protocol: rigid body
Refinement space: REAL
Fitting 2
Initial atomic model: 2WWY
Fitting software: Chimera
Fitting protocol: rigid body
Refinement space: REAL
Fitting 3
Initial atomic model: 4bk0
Fitting protocol: rigid body
Refinement space: REAL

Imaging

Session
Microscope model: FEI TECNAI F20
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.20
Nominal magnification: 100000.0
Defocus max (nm): 2,800.00
Defocus min (nm): 1,200.00
Holder model: GATAN LIQUID NITROGEN
Acquisition
Number of images: 5224

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.037
Buffer: 20 mM Hepes pH 8.0, 4mM MgCl2, 50 mM KCl, 0.05% NP-40, 1mM TCEP
Specimen support: 400-mesh C-flats (Protochips Inc.) with thin carbon support (Protochips Inc.)
Vitrification
Apparatus: FEI VITROBOT MARK II
Cryogen: ETHANE
Humidity: 100
Protocol: blot for 2 sec, 0 offset